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

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.

Immunofluorescence analysis of 3D7elo1-pfs16-CBG99 gametocytes with antibodies specific for the Myc tag sequence fused to the CBG99 luciferase and for the gametocyte-specific protein Pfg27.
Magnification bar is 10m. S-6

Supplementary Figure S-5
Bioluminescence emission spectra of the CBG99 (solid line) and CBR (dashed line) luciferases.
The procedure to produce constructs able to mediate the chromosomal integration of the luciferase cassettes was as follows. First, a unique HindIII site in the pDC2 backbone site was disrupted by HindIII digestion and a Klenow fill-in reaction. An attB site was inserted by overlapping PCR between pfelo1 gene homology regions (HRs) as follows. As above, a HindIII site in pfelo1 HR-I was mutated by amplifying HRI using primers #8-pfelo1HR-I-dir and #9-pfelo1-HR-I-rev1, ligating it into pGEM-T plasmid (Promega). The resulting plasmid was linearised by HindIII digestion, S-9 filled-in with Klenow polymerase, re-ligated and used as template for PCR amplification with above primer #8-pfelo1HR-I-dir and #10-pfelo1-HR-I-rev2. The latter contained, in 5' to 3' direction, stop codon, HindIII and attB sites. HR-II PCR amplification was performed with primers #11-pfelo1-HR-II-dir and #12-pfelo1-HR-II-rev. The former contained, 5' to 3', attB and HindIII sites. HR-I and HR-II PCR products were gel purified and mixed together in a PCR mix lacking primers and subjected to 5 cycles of amplification to generate a full-length HR-I-HindIII-attB-HindIII -HR-II template. Then, above primers #8-pfelo1HR-I-dir and #12-pfelo1-HR-II-rev were added to get the final PCR product, which was digested with ApaI-BamHI, gel purified and inserted into AvrII-BamH I digested pDC22-(mutHindIII) vector, producing the pfelo1-attB donor plasmid.
Digestion of the latter plasmid with HindIII released the attB site which was replaced by the HindIII-HindIII attB-containing luciferase cassette from the pCR2.1 vectors.

Plasmid copy number determination by SYBR-Green Real-time PCR.
The bsd selectable marker (primers #13-bsd-fw and #14-bsd-rev) was used along with the pfeba175 gene (primers #15-pfeba175-fw and #16-pfeba175-rev) as a control for genome numbers. Genomic DNA was extracted from asexual cultures of episomal transfectants expressing the different luciferases alongside with parental 3D7 parasites using the Bioline Blood DNA kit. DNA (50ng in 2l) was added to a 13l PCR mix containing 3.7l water, 7.5l PCR Sybrgreen Master Mix and 0.9l of each primer (final concentration 300nM). Real-time assays were performed using ABI Prism 7500 Real-time PCR System (Applied Biosystems, Foster City, CA) and 7500 Software v2.0.5. The PCR parameters were as follows: 20 sec at 95°C followed by 40 cycles of 95°C for 15 sec and 58°C for 30 sec. Fluorescent product was detected at the last step of each cycle. Plasmid copy numbers were determined against reference amplification titration curves of plasmid (10 2 to 10 7 copies) in 25ng of 3D7 genomic DNA for bsd and of 3D7 genomic DNA (1.25 to 100ng) in 10 7 plasmid copies for eba175. All samples and controls were run in triplicate, normalized as plasmid copy number/genome and expressed as fold variation compared to PpyWT luciferase.