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Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P . falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P . vivax hypnozoites and complete maturation of P . vivax and P . falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research. Currently available platforms to study liver stage of Plasmodium species have limitations. Here, the authors show that primary human hepatocyte cultures in 384-well format support hypnozoite and other liver stage development and are suitable for drug and antibody screens.

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Plasmodium falciparum remains a significant global pathogen, causing over 200 million infections and over 600,000 deaths per year. One significant obstacle to the control of malaria is increasing resistance to first-line artemisinin-based antimalarials. Another is a lack of basic knowledge about the cell biology of the parasite. Along with the mitochondrion, Plasmodium contains a second organelle descended from an endosymbiotic event, the apicoplast. Both organelles are common targets for antimalarials, but because many proteins involved in organellar fission are not conserved in Plasmodium , until now, the mechanisms underlying apicoplast and mitochondrial division have been unknown. In this study, we demonstrate that PfDyn2, a dynamin-related protein (DRP), is required for the division of both organelles. We also show that defects in organellar division hinder segmentation of the schizont and formation of invasive merozoites by preventing full contraction of the basal complex. By demonstrating its necessity for the proper division of both the apicoplast and the mitochondria, this study highlights PfDyn2 as a potential target for new antimalarials.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax , and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi . We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria. The lipid kinase phosphatidylinositol-4-OH kinase (PI(4)K) is identified as a target of the imidazopyrazines, a new antimalarial compound class that can inhibit several Plasmodium species at each stage of the parasite life cycle; the imidazopyrazines exert their inhibitory action by interacting with the ATP-binding pocket of PI(4)K. A multifunction target for antimalarials To eliminate malaria completely it is necessary to cure an individual of all stages in the malaria parasite's life cycle including the symptomatic blood-stage infection and the preceding liver-stage infection (to prevent relapse) and also to block transmission to mosquitoes. Here Elizabeth Winzeler and colleagues identify phosphatidylinositol-4-OH kinase (PI(4)K) as a potential drug target that is essential to fatty acid metabolism in all stages of the Plasmodium parasite. The authors show that a family of compounds with an imidazopyrazine core, distinct from known antimalarials, inhibits PI(4)K and also inhibits the development of multiple Plasmodium species at each stage of the life cycle. Their analyses reveal that the imidazopyrazines interact with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4 phosphate and interfering with cell division.

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase β (PDEβ) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEβ-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEβ plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.

Plasmodium vivax and P. falciparum, the parasites responsible for most human malaria worldwide, exhibit striking biological differences, which have important clinical consequences. Unfortunately, P. vivax, unlike P. falciparum, cannot be cultivated continuously in vitro, which limits our understanding of its biology and, consequently, our ability to effectively control vivax malaria. Here, we describe single-cell gene expression profiles of 9,215 P. vivax parasites from bloodstream infections of Aotus and Saimiri monkeys. Our results show that transcription of most P. vivax genes occurs during short periods of the intraerythrocytic cycle and that this pattern of gene expression is conserved in other Plasmodium species. However, we also identify a strikingly high proportion of species-specific transcripts in late schizonts, possibly associated with the specificity of erythrocyte invasion. Our findings provide new and robust markers of blood-stage parasites, including some that are specific to the elusive P. vivax male gametocytes, and will be useful for analyzing gene expression data from laboratory and field samples.

We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum . In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance. The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 . Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum , we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum . In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona , putative Sarcocystis -unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis -unique transcripts will provide insights into the interesting biology of this parasite genus.