National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 113 Thailand Science Park, Pathumthani 12120, Thailand.
Malar J. 2011 May 10;10:119. doi: 10.1186/1475-2875-10-119.
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.
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.
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.
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.
人类疟原虫物种的耐药性的流行通常与寄生虫内的基因组突变有关。当引入新药时，这种现象需要对可能的耐药突变进行进化预测研究。因此，鉴定可能的新疟原虫二氢叶酸还原酶（PfDHFR）突变体，这些突变体对叶酸类抗疟药物具有抗性，对于叶酸类抗疟药物开发过程至关重要。
开发了一种使用动物疟原虫寄生虫模型鉴定赋予抗叶酸药物抗性的 Pfdhfr 基因突变的系统。通过易错 PCR 和疟原虫转染技术，生成 Pfdhfr 突变体文库，然后外源性转染到疟原虫伯氏疟原虫中，从这些寄生虫中选择对氨苯砜具有抗性的 PfDHFR 突变体。
从该实验中发现的主要突变是 S108N，与从田间分离的第一个氨苯砜耐药突变一致。生成了一个内源性 Pbdhfr 等位基因被突变 PfdhfrS108N 取代的转基因 P. berghei，并证实其与亲本非转基因寄生虫相比具有正常的生长速度，并且对氨苯砜具有抗性。
这项研究证明了转基因 P. berghei 系统在体内寄生虫/宿主环境中预测 PfDHFR 耐药突变的能力。该系统可用于鉴定可能出现的新的叶酸类抗疟化合物耐药突变体，并预测耐药突变的进化。
