title: Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening.
abstract: BACKGROUND
  Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites.
  
  
  METHODS
  Plasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.
  
  
  RESULTS
  Transgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.
  
  
  CONCLUSIONS
  With the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.
authors: Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand.
authors: Voravuth
authors: V
authors: Somsak
authors: Chairat
authors: C
authors: Uthaipibull
authors: Parichat
authors: P
authors: Prommana
authors: Somdet
authors: S
authors: Srichairatanakool
authors: Yongyuth
authors: Y
authors: Yuthavong
authors: Sumalee
authors: S
authors: Kamchonwongpaisan
mesh_terms: D000962:Antimalarials / Q000302:isolation & purification* / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005493:Folic Acid Antagonists / Q000302:isolation & purification* / Q000494:pharmacology; D042822:Genomic Instability; D006801:Humans; D030781:Organisms, Genetically Modified; D010962:Plasmodium berghei / Q000201:enzymology* / Q000235:genetics; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D010966:Plasmodium vivax / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000962:Antimalarials; D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase