Abstract
BackgroundThe 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.MethodsA 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.ResultsThe 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.ConclusionThis 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.
Highlights
The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites
The accumulation of point mutations in Pfdhfr reduces the affinity of antifolate drugs such as pyrimethamine for the enzyme leading to drug resistance [5,6]
Despite the emergence of resistant Pfdhfr mutants in malaria endemic areas, the elucidation and development of target based screening models [8,9] and the solution of the crystal structure of PfDHFRTS [10] means that this enzyme is still an attractive target for drug development
Summary
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. 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. Mutations in Pfdhfr gene, associated with the amino acid substitution at residues 51, 59, 108, and 164, have been found in the field with different levels of resistance to antifolate drugs [3,4] These mutations are positioned around the enzyme active site. The accumulation of point mutations in Pfdhfr reduces the affinity of antifolate drugs such as pyrimethamine for the enzyme leading to drug resistance [5,6]. In order to develop new compounds against drug resistant parasites, prediction of possible future resistance mutations becomes a priority
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