Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum

Abstract

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.

Fig. 1. Experimental scheme of the functional screening approach used to identify a drug-resistance gene from a single drug-resistant P. falciparum strain.

High-coverage genomic libraries were generated in drug-sensitive parasites (blue) from drug-resistant strains (yellow) using the centromere plasmid pFCENv1. The recipient parasite acquired drug resistance due to the introduction of the drug-resistance gene; thus, it survived during drug screening. The DNA fragment inserted in pFCENv1 was recovered from the surviving parasites, and its sequence was determined using genome-walking analysis.

Fig. 2. Functional screening of a chloroquine-resistance gene from the genomic libraries of strain Dd2.

a Parasites [dd2-lib1 (red), 2 (blue), 3D7 (black), Dd2 (orange), and the negative control parasite (yellow)] were treated with 20-nM chloroquine for 6 days. The transgenic parasite, in which the pFCENv1 was introduced, was used as a negative control. b The IC₅₀ values of clonal parasites selected from dd2-lib1 and 2 were determined from The values for strains 3D7 and Dd2 and the negative control parasite was also determined. All assays were performed using n = 4 biological independent samples. Error bars are SEM. The color specification is the same as that used in a. c Genomic DNA fragments were obtained from surviving parasites in dd2-lib1 and 2. DNA fragments overlapped at the genomic region encoding the pfcrt gene. Source data are provided as a Source Data file.

Fig. 3. Functional screening of a mefloquine-resistance gene from the genomic libraries of strain MEF1.

a Parasites in mef-lib3 (red), and 6 (blue), 3D7 (black), MEF1 (orange), and the negative control parasite (yellow) were treated for 6 days with 15-nM mefloquine. b Four genomic DNA fragments were recovered from the parasites, which were selected from mef-lib3, 6, 10, and 13. c The IC₅₀ values of clonal parasites selected from mef-lib3, mef-lib-6, 3D7, MEF1, and the negative control parasite were determined. All assays were performed using n = 4 biological independent samples. Error bars are SEM. The color specification is the same as that used in a. Source data are provided as a Source Data file.

Fig. 4. Mefloquine resistance caused by PfMDR7.

a The mefloquine resistance of a transgenic parasite, into which pfmdr7 of the MEF1 (red) and the 3D7 (blue) were introduced, were examined. In addition, the mefloquine resistance of the 3D7 (black), the MEF1 (orange), and the negative control parasite (yellow) into which only pFCENv1 was introduced were also examined. b The mRNA level of pfmdr7 in the MEF1(orange) was analyzed by RT-qPCR. In addition, those of three mefloquine-sensitive (yellow) and two mefloquine-resistant parasites (red), which were collected from patients living in the Thai–Myanmar border area, were analyzed. Strains 3D7 (green) were used as a negative control. All assays were performed using n = 3 biological independent samples of each parasite strains. Error bars are SEM, and the measure of the center is the means value. The P values were calculated from statistical analysis with a two-sided Student’s t test. Asterisks indicate P values <0.05 for comparisons between the 3D7 and the resistant parasites. c The promoter activities of the pfmdr7 of the MEF1 (red) and the 3D7 (bule) were examined using the transgenic parasites, in which the plasmids having each promoter were introduced. Transfection was performed in duplicates for each plasmid. The relative changes of luminescence of transgenic parasites were estimated based on one transgenic parasite, in which the promoter of pfmdr7 of the 3D7 was introduced. Assays were performed using n = 3 biological independent samples of transgenic parasites. Error bars are SEM, and the measure of the center is the means value. The P values were calculated from statistical analysis with a two-sided Student’s t test. Asterisks indicate P values <0.005 for comparison between the samples. Source data are provided as a Source Data file.