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Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites

[Image: see text] 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....

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Autores principales: Cevenini, Luca, Camarda, Grazia, Michelini, Elisa, Siciliano, Giulia, Calabretta, Maria Maddalena, Bona, Roberta, Kumar, T. R. Santha, Cara, Andrea, Branchini, Bruce R., Fidock, David A., Roda, Aldo, Alano, Pietro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151787/
https://www.ncbi.nlm.nih.gov/pubmed/25102353
http://dx.doi.org/10.1021/ac502098w
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author Cevenini, Luca
Camarda, Grazia
Michelini, Elisa
Siciliano, Giulia
Calabretta, Maria Maddalena
Bona, Roberta
Kumar, T. R. Santha
Cara, Andrea
Branchini, Bruce R.
Fidock, David A.
Roda, Aldo
Alano, Pietro
author_facet Cevenini, Luca
Camarda, Grazia
Michelini, Elisa
Siciliano, Giulia
Calabretta, Maria Maddalena
Bona, Roberta
Kumar, T. R. Santha
Cara, Andrea
Branchini, Bruce R.
Fidock, David A.
Roda, Aldo
Alano, Pietro
author_sort Cevenini, Luca
collection PubMed
description [Image: see text] 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.
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spelling pubmed-41517872015-08-07 Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites Cevenini, Luca Camarda, Grazia Michelini, Elisa Siciliano, Giulia Calabretta, Maria Maddalena Bona, Roberta Kumar, T. R. Santha Cara, Andrea Branchini, Bruce R. Fidock, David A. Roda, Aldo Alano, Pietro Anal Chem [Image: see text] 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. American Chemical Society 2014-08-07 2014-09-02 /pmc/articles/PMC4151787/ /pubmed/25102353 http://dx.doi.org/10.1021/ac502098w Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html)
spellingShingle Cevenini, Luca
Camarda, Grazia
Michelini, Elisa
Siciliano, Giulia
Calabretta, Maria Maddalena
Bona, Roberta
Kumar, T. R. Santha
Cara, Andrea
Branchini, Bruce R.
Fidock, David A.
Roda, Aldo
Alano, Pietro
Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
title Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
title_full Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
title_fullStr Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
title_full_unstemmed Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
title_short Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites
title_sort multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in plasmodium falciparum parasites
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151787/
https://www.ncbi.nlm.nih.gov/pubmed/25102353
http://dx.doi.org/10.1021/ac502098w
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