title: Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.
abstract: New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.
authors.affiliation: INBB, Istituto Nazionale di Biostrutture e Biosistemi , 00136 Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Cevenini
authors.forename: Grazia
authors.initials: G
authors.lastname: Camarda
authors.forename: Elisa
authors.initials: E
authors.lastname: Michelini
authors.forename: Giulia
authors.initials: G
authors.lastname: Siciliano
authors.forename: Maria Maddalena
authors.initials: MM
authors.lastname: Calabretta
authors.forename: Roberta
authors.initials: R
authors.lastname: Bona
authors.forename: T R Santha
authors.initials: TR
authors.lastname: Kumar
authors.forename: Andrea
authors.initials: A
authors.lastname: Cara
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Branchini
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
authors.forename: Pietro
authors.initials: P
authors.lastname: Alano
mesh_terms: D000962:Antimalarials / Q000494:pharmacology; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D018715:Microscopy, Video; D010274:Parasitology / Q000379:methods*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics; D059010:Single-Cell Analysis
chemical_list: D000962:Antimalarials; D015800:Protozoan Proteins; D008156:Luciferases
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