Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

Abstract

Background: The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.

Methods: A system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected.

Results: The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.

Conclusion: This study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.

Figure 1

Sensitivity of pyrimethamine against PbGFP parasite. The data represents mean values ± SD of percentage of growth inhibition for 5 animals per group. The average ED₅₀ from three independent studies is 0.02 mg/kg.

Figure 2

Replacement strategy of mutant Pfdhfr-ts into Pbdhfr-ts by double cross-over homologous recombination. (A) endogenous Pbdhfr-ts gene locus in PbGFP wild-type parasite, (B) linearized plasmid pY005 containing mutant Pfdhfr^S108N, (C) Pfdhfr^S108N-ts gene locus in transgenic PbPfS108N parasite. Positions of primers used for PCR amplification are indicated by arrows. The expected size of PCR products are described.

Figure 3

PCR and RT-PCR analysis of the transgenic mutant parasite. (A) PCR analysis of 5' and 3' integrations on genomic DNA isolated from transgenic mutant parasites, PbPfS108N, are shown in lanes 1 and 4, respectively. Genomic DNA isolated from PbGFP wild-type parasite and distilled water (Neg) served as negative controls as shown in lanes 2, 5 and 3, 6, respectively. (B) RT-PCR analysis of PbPfS108N parasites. RNA from the PbPfS108N parasite was reverse transcribed to cDNA and used as a template to amplify Pfdhfr, Pbdhfr and P. berghei alpha tubulin (Pbα-tubulin) genes. The reactions were performed with reverse transcription (lane 1), without reverse transcription (lane 2), P. berghei cDNA derived from PbGFP and distilled water (Neg) were used as negative controls with and without reverse transcription (lanes 3-6).

Figure 4

Growth rate and pyrimethamine sensitivity of PbPfS108N parasite. (A) Growth curves of PbPfS108N and PbGFP in mice. PbPfS108N data are represented as open circles and, PbGFP data represented as filled circles. The experiments were performed in three independent studies and the data represents mean ± SD values. (B) Sensitivity of pyrimethamine against PbPfS108N parasite. The data represents mean ± SD of percentage of growth inhibition. The average ED₅₀ from three independent studies is 1.1 mg/kg.