Na+ Influx Induced by New Antimalarials Causes Rapid Alterations in the Cholesterol Content and Morphology of Plasmodium falciparum

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Abstract
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. Malaria remains a major public health challenge in the world, especially with the realization that parasites causing the disease are becoming resistant to currently used antimalarial drugs. There are new antimalarial drugs under development, and among these are 3 clinical candidate drugs that have the propensity to cause sodium leakage into parasites growing inside human red blood cells. We have investigated events that lead to parasite death when sodium concentration inside the parasite increases. Our findings suggest that the drug-treated parasite rapidly acquires cholesterol and clusters containing lipid-embedded proteins MSP1 and MSP2 form within the plasma membrane. Because these changes are reversible when the drugs are removed, we suggest that there is an active process that keeps cholesterol out of the parasite. We also observed massive morphological changes resembling premature steps of parasite division following drug treatment. The changes we describe appear to be a direct consequence of increased sodium level in the parasites. We hypothesize that sodium influx constitutes a normal signaling process in malaria parasites and that the new antimalarial drugs initiate this process prematurely, which results in the death of the parasites.
Funding Information
  • National Institutes of Health (AI098413)

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