Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Antirelapse Compounds.

Annemarie M Voorberg-Van Der Wel,Onny Klop,Clemens H M Kocken,Ivonne G Nieuwenhuis,Anne-Marie Zeeman,Lars C Vermaat,Els J Klooster,Nicole M Van Der Werff

doi:10.1021/acs.analchem.0c00547

Annemarie M Voorberg-Van Der Wel, Onny Klop + Show 6 more

Open Access

https://doi.org/10.1021/acs.analchem.0c00547

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Journal: Analytical chemistry	Publication Date: Apr 8, 2020
Citations: 8	License type: cc-by

Affiliation: Biomedical Primate Research Centre

Abstract

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.

Highlights

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease
In contrast to the other major malaria parasite P. falciparum, P. vivax can develop into hypnozoites, persisting forms in the liver that can reactivate after prolonged periods of time, to give rise to new transmissible stages and to cause new episodes of malaria.[5,6]
Notwithstanding these challenges, much progress has been made over the past few years, resulting in different in vitro liver stage platforms in which compounds can be tested for activity against hypnozoites.[10−13] The read-out of these assays relies on high-content fluorescence imaging to distinguish small from large forms, which limits the throughput of the assay because of the timeconsuming imaging and analysis

Summary

Introduction

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Cynomolgi liver stages, the Lisp[2] protein is expressed in multinucleate parasites, starting at the onset of liver stage schizogony, 3 days post sporozoite infection.[25] A similar stagespecific expression was observed in a P. cynomolgi fluorescent reporter line that showed schizont-specific expression of mCherry controlled by lisp[2] flanking regions.[26] This reporter line, which shows a fitness comparable to wildtype parasites,[26] contains the bioluminescent reporter Nluc linked by T2A33 with mCherry under control of lisp2.26 We here use this single bioluminescent ‘Sz_Nluc’ line as a reference for the dual ‘All_Nluc’ line which contains the Fluc bioluminescent reporter driven by lisp[2] (Figure 1a) to control for possible confounding data arising from a previously described 150-fold lower brightness of Fluc compared to Nluc.[23,31] To characterize the temporal kinetics of the bioluminescent signals of the dual reporter throughout liver stage development, a time course experiment was performed.

Results

Conclusion