Detailing organelle division and segregation in Plasmodium falciparum.

The malaria causing parasite, Plasmodium falciparum, replicates through a tightly orchestrated process termed schizogony, where approximately 32 daughter parasites are formed in a single infected red blood cell and thousands of daughter cells in mosquito or liver stages. One-per-cell organelles, such as the mitochondrion and apicoplast, need to be properly divided and segregated to ensure a complete set of organelles per daughter parasites. Although this is highly essential, details about the processes and mechanisms involved remain unknown. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D-imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during the last stage of schizogony. Analysis of focused ion beam scanning electron microscopy (FIB-SEM) data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques (CPs) in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation.

Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183.

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (AcAS) inhibitor to enter preclinical development. Our studies demonstrate attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound shows single digit nanomolar in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocks P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identify AcAS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. Pharmacokinetic-pharmacodynamic modelling predict that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. Toxicology studies in rats indicate a > 30-fold safety margin in relation to the predicted human efficacious exposure. In conclusion, MMV693183 represents a promising candidate for further (pre)clinical development with a novel mode of action for treatment of malaria and blocking transmission.

RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes.

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.

Organelle Dynamics in Apicomplexan Parasites.

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium falciparum, are the cause of many important human and animal diseases. While T. gondii tachyzoites replicate through endodyogeny, during which two daughter cells are formed within the parental cell, P. falciparum replicates through schizogony, where up to 32 parasites are formed in a single infected red blood cell and even thousands of daughter cells during mosquito- or liver-stage development. These processes require a tightly orchestrated division and distribution over the daughter parasites of one-per-cell organelles such as the mitochondrion and apicoplast. Although proper organelle segregation is highly essential, the molecular mechanism and the key proteins involved remain largely unknown. In this review, we describe organelle dynamics during cell division in T. gondii and P. falciparum, summarize the current understanding of the molecular mechanisms underlying organelle fission in these parasites, and introduce candidate fission proteins.

Antimalarial pantothenamide metabolites target acetyl-coenzyme A biosynthesis in Plasmodium falciparum.

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl-coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl-coenzyme A synthetase and acyl-coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.