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.

Luciferase-Based, High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes

“…Compounds belonging to the 4-aminoquinoline class are known to be potent asexual-and early gametocyte stage inhibitors (15). Their activity has been reported to drop as gametocytogenesis progresses (16,19,31). Indeed, all 4-aminoquinolines tested (AQ-13, chloroquine, hydroxychloroquine, amodiaquine, naphthoquine, and piperaquine) failed to inhibit late-stage gametocytes in our assay.…”

A high susceptibility to redox imbalance of the transmissible stages of Plasmodium falciparum revealed with a luciferase‐based mature gametocyte assay

“…Gametocytes were dispensed as described above with 1 μM of the different compounds and incubated with MB at 37°C. Cell viability was evaluated by adding a non lysing formulation of 0.5 mM D-Luciferin substrate (Cevenini et al ., 2014) and measuring luciferase activity for 1 second on a Varioskan™ Flash Multimode Reader (Thermo Scientific). The percent viability was calculated as a function of drug concentration.…”

Ensuring transmission through dynamic host environments: host–pathogen interactions in Plasmodium sexual development

“…Some of these assays rely on fluorescent or luminescent reporters, enabling monitoring of gametocyte-specific drug activity [47,48], while others use DNA dyes, viability dyes, or enzymatic assays to allow screening of all parasite lines including field isolates [49–52]. In addition, new readouts for transmission-blocking activity [53,54] will increase throughput of drug and vaccine testing while the sex-specific proteome of mature gametocytes [55] may help identify stage-specific biomarkers.…”