Abstract
BackgroundPlasmodium 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.MethodsPlasmodium 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.ResultsTransgenic 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.ConclusionWith 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.
Highlights
Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent
Chloroquine has been used as the standard treatment for blood stage vivax malaria for more than 40 years; chloroquine-resistant P. vivax has been reported in many parts of the world [3,4,5]
Generation of transgenic Plasmodium falciparum harbouring Pvdhfr-ts gene Pyrimethamine-resistant P. falciparum strain K1CB1 was transfected with plasmid DNA for replacement of the endogenous Pfdhfr-ts with wild-type Pvdhfr-ts
Summary
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. Current anti-malarial drug development efforts are focused on screening for lead compounds against Plasmodium falciparum, the most lethal species. Plasmodium falciparum can be routinely cultured in vitro and is amenable to high throughput compound screening. Anti-malarial drug development against other human Plasmodium species, Plasmodium vivax - which is the most prevalent cause of human malaria in tropical regions, with an estimated 80 million cases annually [1], is neglected in comparison, since continuous in vitro culture methods are not available for these species. There is an urgent need for new anti-vivax anti-malarial drugs
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