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.

Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging inPlasmodium falciparumParasites / 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. - In: ANALYTICAL CHEMISTRY. - ISSN 0003-2700. - STAMPA. - 86:(2014), pp. 8814-8821. [10.1021/ac502098w]

Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging inPlasmodium falciparumParasites

CEVENINI, LUCA;MICHELINI, ELISA;CALABRETTA, MARIA MADDALENA;RODA, ALDO;
2014

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.
2014
Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging inPlasmodium falciparumParasites / 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. - In: ANALYTICAL CHEMISTRY. - ISSN 0003-2700. - STAMPA. - 86:(2014), pp. 8814-8821. [10.1021/ac502098w]
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
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/393659
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