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

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.

Evidence for Pyronaridine as a Highly Effective Partner Drug for Treatment of Artemisinin-Resistant Malaria in a Rodent Model

“…Prior studies have shown that P. berghei and P. falciparum can share the same genetic basis of resistance to a number of antimalarial drugs, including pyrimethamine, atovaquone, and MFQ (67)(68)(69)(70)(71)(72). The same is not true for chloroquine however, which in the case of P. berghei and other rodent parasites has as-yet undefined mechanisms of resistance that do not appear to involve orthologs of pfcrt (64,73).…”

Developmental Sensitivity in Schistosoma mansoni to Puromycin To Establish Drug Selection of Transgenic Schistosomes

“…Challenges such as reaching effective concentrations of the antibiotic in serum to select transgenic schistosomes in vivo might be encountered. However, at the present time, similar approaches are routinely applied to select transgenic protozoan parasites (56)(57)(58). Moreover, schistosomicidal drugs, including oxamniquine and praziquantel, have been employed to treat infected snails (59), paving the way for similar approaches with antibiotics, e.g., puromycin and neomycin.…”

Development of Plasmodium falciparum Protease Inhibitors in the Past Decade (2002–2012)

“…To the first target category belong Pf enzymes in charge of generating nutrients required for malaria parasite growth. For instance, there are different biosynthesis pathways today used as targets for the discovery of antimalarial drugs such as the folate biosynthesis pathway [11][12][13]. The second target category includes pathways which mediate the uptake of nutrients into cells and the generation and maintenance of transmembrane electrochemical gradients, for instance, the plasmodial surface anion channel (PSAC) [14][15][16].…”