Date Published: May 26, 2016
Publisher: Public Library of Science
Author(s): Sudipta Das, Suyash Bhatanagar, Joanne M. Morrisey, Thomas M. Daly, James M. Burns, Isabelle Coppens, Akhil B. Vaidya, Margaret A Phillips.
Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i) within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2), glycosylphosphotidylinositol (GPI)-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble “rhoptries” and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise.
Billions of people living in regions endemic for malaria are confronted with the looming threat of Plasmodium falciparum parasites resistant to currently effective artemisinin combination therapies . From an evolutionary point of view, emergence of resistant parasites has to be anticipated, especially in light of the fact that the drug pressure is being applied on a vast population of parasites, whose transmission requires obligatory sexual reproduction favoring recombinatorial selection of beneficial drug resistance alleles. Thus for a foreseeable future, efforts to control and eliminate malaria will require a robust pipeline of antimalarial drugs under development. Over the past decade, efforts by academic and industrial investigators have begun to prime this pipeline with new chemical entities with potent antimalarial activity . Understanding the mechanism by which these new compounds cause the demise of malaria parasites would reveal vulnerable aspects of parasite physiology, which in turn could identify other new potential drug targets for further investigations.
The results above provide a glimpse of a complex set of events that are triggered by an influx of Na+ into the cytoplasm of trophozoite stages of P. falciparum. By using a simple saponin sensitivity assay, we have demonstrated profound changes in plasma membrane permeability of the trophozoite stage P. falciparum induced by two chemically distinct antimalarial drugs. We recognize that this approach does not directly measure the parasite plasma membrane properties. However, the membrane organization of intraerythrocytic parasites makes it technically very difficult, if not impossible, to separate PPM from PVM to carry out direct biochemical assessment of their content. Close proximity of the two membranes also makes it very difficult to use fluorescent probes to monitor their relative cholesterol contents. The approach of fluorescence lifetime imaging microscopy to assess cholesterol content of PPM vs. PVM does not provide adequate resolution in live imaging of parasites and thus is not suitable for this purpose . Thus, we have relied on the well-documented propensity of saponin (and digitonin) to insert pores within membranes in a cholesterol-dependent manner, leading to the leakage of cytosolic proteins, to provide evidence for dramatic but reversible changes in the cholesterol content of the PPM induced by these antimalarial drugs. Experiments described in Fig 3, in which MβCD and MβCD loaded with cholesterol were used prior to saponin sensitivity assessment of treated and untreated freed parasites, provide strong support to our interpretations of rapid accumulation of cholesterol in the PPM by drug treated parasites.