Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening

Malaria Journal

Methodology

Somdet Srichairatanakool¹  Chairat Uthaipibull²  Sumalee Kamchonwongpaisan²  Parichat Prommana²  Yongyuth Yuthavong²  Voravuth Somsak³ 

[1] Department of Biochemistry, Faculty of Medicine, Chiang Mai University, 50200, Chiang Mai, Thailand;Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, 12120, Pathumthani, Thailand;Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, 12120, Pathumthani, Thailand;Department of Biochemistry, Faculty of Medicine, Chiang Mai University, 50200, Chiang Mai, Thailand;

关键词: Plasmodium;    Artesunate;    Pyrimethamine;    Plasmodium Berghei;    Parasite Line;   

DOI  :  10.1186/1475-2875-10-291

 received in 2011-08-17, accepted in 2011-10-07,  发布年份 2011

来源: Springer

【 摘 要 】

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 Pv DHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type Pv DHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant Pv DHFR-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 Pv DHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting Pv DHFR-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.

【 授权许可】

Unknown   
© Somsak et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.