Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
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- 作者：Luca Cevenini ; Grazia Camarda ; Elisa Michelini ; Giulia Siciliano ; Maria Maddalena Calabretta ; Roberta Bona ; T. R. Santha Kumar ; Andrea Cara ; Bruce R. Branchini ; David A. Fidock ; Aldo Roda ; Pietro Alano
- 刊名：Analytical Chemistry
- 出版年：2014
- 出版时间：September 2, 2014
- 年：2014
- 卷：86
- 期：17
- 页码：8814-8821
- 全文大小：400K
- ISSN：1520-6882
文摘
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鈥?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鈥?factor of 0.71 卤 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.