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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.

SUBMITTER: Cevenini L

PROVIDER: S-EPMC4151787 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r2 for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86 k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r2, 0.87-0.96), but the performance of IMPUTE was mixed (allelic r2, 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima's D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and association analyses, and supporting global surveillance for drug resistance markers and candidate vaccine antigens.

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Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

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