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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.

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.

UDP-N-acetylglucosamine (UDP-GlcNAc) is a crucial sugar nucleotide for glycan synthesis in eukaryotes. In the malaria parasite Plasmodium falciparum , UDP-GlcNAc is synthesized via the hexosamine biosynthetic pathway (HBP) and is essential for glycosylphosphatidylinositol (GPI) anchor production, the most prominent form of protein glycosylation in the parasite. In this study, we explore a conditional knockout of glucosamine-6-phosphate N-acetyltransferase ( Pf GNA1), a key HBP enzyme. Pf GNA1 depletion led to significant disruptions in HBP metabolites, impairing GPI biosynthesis and causing mislocalization of the merozoite surface protein 1 (MSP1), the most abundant GPI-anchored protein in the parasite. Furthermore, parasites were arrested at the schizont stage, exhibiting severe segmentation defects and an incomplete rupture of the parasitophorous vacuole membrane (PVM), preventing egress from host red blood cells. Our findings demonstrate the critical role of HBP and GPI biosynthesis in P. falciparum asexual blood stage development and underscore the potential of targeting these pathways as a therapeutic strategy against malaria.

Background Plasmodium falciparum merozoite invasion of erythrocytes is an essential step in the asexual blood-stage cycle and a major target for antimalarial intervention. Rhoptry neck proteins play key roles in the formation and function of the tight junction, yet many remain poorly characterized. RALP1, a conserved rhoptry neck-associated leucine zipper-like protein, has been proposed to participate in erythrocyte binding and invasion. Conventional gene disruption attempts have been unsuccessful, suggesting that RALP1 may be essential for parasite survival. Nevertheless, its precise role and broader molecular impact during intraerythrocytic development remain to be fully elucidated. Methods We generated a 3 × HA-tagged conditional knockdown line ( ralp1-ha-glmS ) using CRISPR-Cas9-mediated homologous recombination. RALP1 abundance and subcellular localization were evaluated by Western blotting and immunofluorescence assays. Effects on parasite growth, schizont maturation, merozoite invasion, and merozoite numbers were assessed using tightly synchronized cultures and established invasion and cytological assays. Transcriptomic changes following GlcN-induced RALP1 knockdown were analyzed by RNA-seq at early ring and schizont stages. Sequence-based structural and epitope features were examined using IUPred2A, ANCHOR2, AlphaFold3, NetMHCpan, and NetMHCIIpan. Results Precise integration of the ha-glmS cassette enabled GlcN-inducible reduction of RALP1 protein levels, most prominently in schizonts. RALP1 knockdown reduced parasite proliferation, impaired schizont maturation, decreased merozoite numbers, and lowered erythrocyte invasion efficiency. RNA-seq showed limited effects in early rings but widespread downregulation of invasion- and host-parasite interaction-related genes in schizonts after correction for glucosamine-responsive transcripts, with GO enrichment highlighting processes related to host cell interaction, biological adhesion, and membrane-associated components. Sequence-based analyses indicated that RALP1 contains extensive intrinsically disordered regions with multiple predicted interaction motifs, while predicted B- and T-cell epitope hotspots concentrated within the C-terminal RBC-binding domain. AlphaFold3 modeling yielded low global confidence (pTM = 0.23), consistent with a primarily disordered architecture. Conclusions RALP1 is required for normal schizont maturation and efficient erythrocyte invasion in P. falciparum . Its partial knockdown perturbs transcription of key invasion ligands and apical components, indicating a broader role in preparing merozoites for host-cell entry. The extensive disorder, epitope-rich C-terminal region, and essential function of RALP1 highlight its potential as a candidate for therapeutic or vaccine targeting. Graphical Abstract

Novel vaccines are urgently needed to reduce the burden of severe malaria. Using a differential whole-proteome screening method, we identified Plasmodium falciparum schizont egress antigen-1 (PfSEA-1), a 244-kilodalton parasite antigen expressed in schizont-infected red blood cells (RBCs). Antibodies to PfSEA-1 decreased parasite replication by arresting schizont rupture, and conditional disruption of PfSEA-1 resulted in a profound parasite replication defect. Vaccination of mice with recombinant Plasmodium berghei PbSEA-1 significantly reduced parasitemia and delayed mortality after lethal challenge with the Plasmodium berghei strain ANKA. Tanzanian children with antibodies to recombinant PfSEA-1A (rPfSEA-1A) did not experience severe malaria, and Kenyan adolescents and adults with antibodies to rPfSEA-1A had significantly lower parasite densities than individuals without these antibodies. By blocking schizont egress, PfSEA-1 may synergize with other vaccines targeting hepatocyte and RBC invasion.

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.

Upon transmission to the liver, Plasmodium vivax parasites form replicating schizonts, which progress to initiate blood-stage infection, or dormant hypnozoites that reactivate weeks to months after initial infection. P. vivax phenotypes in the field vary significantly, including the time to, and frequency of, relapse. Current evidence suggests that both parasite genetics and environmental factors underly this heterogeneity. Here, we applied an approach called kinase regression to evaluate the extent to which P. vivax liver-stage parasites are susceptible to changes in host kinase activity. We identified a role for a subset of host kinases in regulating the numbers of schizonts and hypnozoites, as well as schizont size, and characterized overlap as well as variability in host phosphosignaling dependencies between parasite forms across multiple patient isolates. Our data point to variability in host dependencies across P. vivax isolates, suggesting one possible origin of the heterogeneity observed in the field.

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.

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

Background Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. Methods HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector ® electroporation technology. Results Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherry Hsp70 -expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. Conclusion An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8–18 days.