Abstract
Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.
Highlights
Malaria is a life-threatening disease caused by five species of parasites belonging to the genus Plasmodium, and includes P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi
With both firefly luciferase and Nluc under control of the same Hsp86 promoter, the plasmids were electroporated into uninfected red blood cells (RBCs) and fed to P. falciparum trophozoite stage cultures so that the parasites would take up the plasmid when they invaded the electroporated RBCs
The so-called transient transfectants were assayed for luciferase activity after saponin lysis which selectively permeabilises the RBC to facilitate the removal of haemoglobin, a powerful quencher of emitted light
Summary
Malaria is a life-threatening disease caused by five species of parasites belonging to the genus Plasmodium, and includes P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. The structural family to which some compounds belong may indicate their likely targets, for most compounds the targets are completely unknown This is further exacerbated by the fact that even if the targets could be identified by complex and costly laboratory investigation, the functions of more than half of the parasite’s proteins are still unknown. This makes it very difficult to deduce what a compound’s mechanism of action might be, making it problematic to evaluate if the compound is worth further development
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