Presentation of a versatile Plasmodium falciparum dual reporter line, expressing both a fluorescent protein and NanoLuc under a constitutive promoter, that can be used to screen for novel anti... Show morePresentation of a versatile Plasmodium falciparum dual reporter line, expressing both a fluorescent protein and NanoLuc under a constitutive promoter, that can be used to screen for novel anti-malarial drugs effective against multiple stages of the parasite.Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages. Show less
Winkel, B.M.F.; Korne, C.M. de; Oosterom, M.N. van; Staphorst, D.; Meijhuis, M.; Baalbergen, E.; ... ; Roestenberg, M. 2019
Given the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can... Show moreGiven the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can fully protect malaria-naive individuals. Despite the fact that motility of these attenuated parasites is key to their infectivity and ultimately protective efficacy, sporozoite motility in human tissue (e.g. skin) remains wholly uncharacterized to date. We show that the ability to quantitatively address the complexity of sporozoite motility in human tissue provides an additional tool in the development of attenuated sporozoite vaccines. We imaged Pf movement in the skin of its natural host and compared wild-type and radiation-attenuated GFP-expressing Pf sporozoites. Using custom image analysis software and human skin explants we were able to quantitatively study their key motility features. This head-to-head comparison revealed that radiation attenuation impaired the capacity of sporozoites to vary their movement angle, velocity and direction, promoting less refined movement patterns. Understanding and overcoming these changes in motility will contribute to the development of an efficacious attenuated parasite malaria vaccine. Show less
Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of... Show moreGenetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37 degrees C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines. Show less
