SummaryThe malaria parasite Plasmodium falciparum induces a sixfold increase in the phospholipid content of infected erythrocytes during its intraerythrocytic growth. We have characterized the lipid environments in parasitized erythrocyte using the hydrophobic probe, Nile Red. Spectral imaging with a confocal microscope revealed heterogeneous lipid environments in parasite-infected erythrocytes. An insight into the nature of these environments was gained by comparing these spectra with those of triacylglycerol/ phospholipid emulsions and phospholipid membranes. Using this approach, we identified a population of intensely stained particles of a few hundred nanometers in size that are closely associated with the digestive vacuole of the parasite and appear to be composed of neutral lipids. Electron microscopy and isolation of food vacuoles confirmed the size of these particles and their intimate association respectively. Lipid analysis suggests that these neutral lipid bodies are composed of di-and triacylgycerols and may represent storage organelles for lipid intermediates that are generated during digestion of phospholipids in the food vacuole. Mono-, di-and triacylglycerol suspensions promote b b b b -haematin formation, suggesting that these neutral lipid bodies, or their precursors, may also be involved in haem detoxification. We also characterized other compartments of the infected erythrocyte that were stained less intensely with the Nile Red probe. Both the erythrocyte membrane and the parasite membrane network exhibit red shifts compared with the neutral lipid bodies that are consistent with cholesterol-rich and cholesterol-poor membranes respectively. Ratiometric imaging revealed more subtle variations in the lipid environments within the parasite membrane network.

Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å electron cryo-microscopy structure of the Plasmodium falciparum 80S-ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated center. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-EM to guide the development of mefloquine derivatives to inhibit parasite protein synthesis. *Correspondence and requests for materials should be addressed to J.B. (jake.baum@imperial.ac.uk) or S.H.W.S. (scheres@mrc-lmb.cam.ac.uk). Author Contributions: W.W., X-C.B., B.E.S., K.E.J, T.T., D.S.M., S.A.R, S.H.W.S and J.B. designed all experiments; W.W., X-C.B, B.E.S., K.E.J, A.B., T.T., D.S.M., J.K.T., E.H. and I.S.F. performed experiments; W.W., X-C.B, B.E.S., K.E.J., T.T., A.B., J.K.T., S.A.R., A.F.C., S.H.W.S. and J.B. contributed to manuscript preparation.Author Information: Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank with accession numbers XXX. Atomic coordinates have been deposited in the Protein Data Bank, with entry codes XXX.The authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nat Microbiol. Author manuscript; available in PMC 2017 September 13. Malaria is a major protozoan parasitic disease that inflicts an enormous burden on global human health. In 2015 the disease resulted in an estimated 429,000 deaths with several hundreds of millions of people infected 1. The causative agents of malaria are a group of protozoan parasites that belong to the genus Plasmodium, a member of the ancient apicomplexan phylum of vertebrate pathogens, with P. falciparum and P. vivax being responsible for the majority of disease mortality and morbidity, respectively 2.Antimalarial chemotherapies have long been the gold-standard utility for the prevention and treatment of malaria. Over many decades, many different classes of antimalarials have been clinically approved and deployed as frontline treatments to combat malaria 3. Despite the long-standing usage of these drugs, their mode of actions in mediating parasite killing are not well defined. Mefloquine (MFQ) has been one of the most effective antimalarials since it was first developed and has been used as a chemoprophylactic drug by visitors staying in malaria endemic area...

Graphical abstract

Plasmodium falciparum develops within the mature RBCs (red blood cells) of its human host in a PV (parasitophorous vacuole) that separates the host cell cytoplasm from the parasite surface. The pore-forming toxin, SLO (streptolysin O), binds to cholesterol-containing membranes and can be used to selectively permeabilize the host cell membrane while leaving the PV membrane intact. We found that in mixtures of infected and uninfected RBCs, SLO preferentially lyses uninfected RBCs rather than infected RBCs, presumably because of differences in cholesterol content of the limiting membrane. This provides a means of generating pure preparations of viable ring stage infected RBCs. As an alternative permeabilizing agent we have characterized EqtII (equinatoxin II), a eukaryotic pore-forming toxin that binds preferentially to sphingomyelin-containing membranes. EqtII lyses the limiting membrane of infected and uninfected RBCs with similar efficiency but does not disrupt the PV membrane. It generates pores of up to 100 nm, which allow entry of antibodies for immunofluorescence and immunogold labelling. The present study provides novel tools for the analysis of this important human pathogen and highlights differences between Plasmodium-infected and uninfected RBCs.