SummaryThe antimalarial spiroindolones disrupt Plasmodium falciparum Na + regulation and induce an alkalinization of the parasite cytosol. It has been proposed that they do so by inhibiting PfATP4, a parasite plasma membrane P-type ATPase postulated to export Na + and import H + equivalents. Here, we screened the 400 antiplasmodial compounds of the open access 'Malaria Box' for their effects on parasite ion regulation. Twenty eight compounds affected parasite Na + and pH regulation in a manner consistent with PfATP4 inhibition. Six of these, with chemically diverse structures, were selected for further analysis. All six showed reduced antiplasmodial activity against spiroindolone-resistant parasites carrying mutations in pfatp4. We exposed parasites to incrementally increasing concentrations of two of the six compounds and in both cases obtained resistant parasites with mutations in pfatp4. The finding that diverse chemotypes have an apparently similar mechanism of action indicates that PfATP4 may be a significant Achilles' heel for the parasite.

BackgroundThe development of differentiated sexual stages (gametocytes) within human red blood cells is essential for the propagation of the malaria parasite, since only mature gametocytes will survive in the mosquito’s midgut. Hence gametocytogenesis is a pre-requisite for transmission of the disease. Physiological changes involved in sexual differentiation are still enigmatic. In particular the lipid metabolism—despite being central to cellular regulation and development—is not well explored.MethodsHere the lipid profiles of red blood cells infected with the five different sexual stages of Plasmodium falciparum were analysed by mass spectrometry and compared to those from uninfected and asexual trophozoite infected erythrocytes.ResultsFundamental differences between erythrocytes infected with the different parasite stages were revealed. In mature gametocytes many lipids that decrease in the trophozoite and early gametocyte infected red blood cells are regained. In particular, regulators of membrane fluidity, cholesterol and sphingomyelin, increased significantly during gametocyte maturation. Neutral lipids (serving mainly as caloriometric reserves) increased from 3 % of total lipids in uninfected to 27 % in stage V gametocyte infected red blood cells. The major membrane lipid class (phospholipids) decreased during gametocyte development.ConclusionsThe lipid profiles of infected erythrocytes are characteristic for the particular parasite life cycle and maturity stages of gametocytes. The obtained lipid profiles are crucial in revealing the lipid metabolism of malaria parasites and identifying targets to interfere with this deadly disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1130-z) contains supplementary material, which is available to authorized users.

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na-dependent ATPase activity present in membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K] or [Ca]. With an apparent for ATP of 0.2 mm and an apparent for Na of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na efflux to increase in response to an increase in cytosolic [Na]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent for Na is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

For an increasing number of antimalarial agents identified in high-throughput phenotypic screens, there is evidence that they target PfATP4, a putative Na efflux transporter on the plasma membrane of the human malaria parasite For several such "PfATP4-associated" compounds, it has been noted that their addition to parasitized erythrocytes results in cell swelling. Here we show that six structurally diverse PfATP4-associated compounds, including the clinical candidate KAE609 (cipargamin), induce swelling of both isolated blood-stage parasites and intact parasitized erythrocytes. The swelling of isolated parasites is dependent on the presence of Na in the external environment and may be attributed to the osmotic consequences of Na uptake. The swelling of the parasitized erythrocyte results in an increase in its osmotic fragility. Countering cell swelling by increasing the osmolarity of the extracellular medium reduces the antiplasmodial efficacy of PfATP4-associated compounds, consistent with cell swelling playing a role in the antimalarial activity of this class of compounds.

Trypanosomatids, which include major pathogens of humans and livestock, are flagellated protozoa for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle defects in Trypanosoma brucei. We induced massive parallel knockdown, sorted the perturbed population using high-throughput flow cytometry, deep-sequenced RNAi-targets from each stage and digitally reconstructed cell cycle profiles at a genomic scale; also enabling data visualisation using an online tool (https://tryp-cycle.pages.dev/). Analysis of several hundred genes that impact cell cycle progression reveals >100 flagellar component knockdowns linked to genome endoreduplication, evidence for metabolic control of the G1-S transition, surface antigen regulatory mRNA-binding protein knockdowns linked to G2M accumulation, and a putative nucleoredoxin required for both mitochondrial genome segregation and for mitosis. The outputs provide comprehensive functional genomic evidence for the known and novel machineries, pathways and regulators that coordinate trypanosome cell cycle progression.