Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.
Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.
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DOI：
10.1021/ac502098w
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发表时间：
2014-09-02
影响因子：
7.4
通讯作者：
Alano, Pietro
中科院分区：
文献类型：
--
作者：
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
迫切需要新的可靠和经济有效的抗疟疾药物筛选试验，以确定作用于恶性疟原虫不同阶段的药物，特别是那些导致人传蚊子的药物，即恶性疟原虫配子体。在目前的配子体分析中，通常报告低Z‘因子、狭窄的动态范围和/或延长的分析时间，以测量配子细胞表达的荧光或荧光素酶报告、内源ATP水平、配子体酶活性或氧化还原依赖的染料荧光。本文报道了恶性疟原虫未成熟和成熟配子体在恶性疟原虫有性期特异性pfs16基因启动子控制下表达红色和绿色荧光素酶的双荧光素酶配子体检测方法。该分析通过参考抗疟疾药物进行了验证，并允许定量和同时测量不同发育阶段的寄生虫特定阶段的药物效果。与最先进的类似分析方法相比，优化的分析方法只需与药物孵育48小时，并使用成本效益高的发光底物，显著降低了分析成本和时间。该方法的Z‘因子为0.71±0.03，适用于96孔板和384孔板。此外，非裂解d-荧光素底物的使用显著提高了分析的可靠性，并首次允许人们在单细胞水平上进行恶性疟原虫生物发光成像。
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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影响因子：
3.7
作者：
Lelièvre J;Almela MJ;Lozano S;Miguel C;Franco V;Leroy D;Herreros E
通讯作者：
Herreros E
影响因子：
3.7
作者：
Helm S;Lehmann C;Nagel A;Stanway RR;Horstmann S;Llinas M;Heussler VT
通讯作者：
Heussler VT
影响因子：
3.4
作者：
Lewis, Michael D.;Francisco, Amanda Fortes;Kelly, John M.
通讯作者：
Kelly, John M.
影响因子：
5.2
作者：
D'Alessandro, Sarah;Silvestrini, Francesco;Taramelli, Donatella
通讯作者：
Taramelli, Donatella
影响因子：
3.6
作者：
Olivieri, Anna;Camarda, Grazia;Alano, Pietro
通讯作者：
Alano, Pietro