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

Somsak V, Uthaipibull C, Prommana P, Srichairatanakool S, Yuthavong Y, Kamchonwongpaisan S - Malar. J. (2011)

Bottom Line: 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.The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

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.

Conclusion: 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.

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Sensitivity of transgenic Plasmodium falciparum expressing wild-type PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and dihydroartemisinin (C). The growth of parasites treated with pyrimethamine, chloroquine and dihydroartemisinin was detected using the SYBR Green I staining assay. The percentage of parasite growth was plotted against drug concentrations. Data were shown as mean ± S.D. of at least 3 independent experiments. K1: P. falciparum K1CB1; TM4: P. falciparum TM4/8.2; B2: transgenic PfPvDTclB2.
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Figure 3: Sensitivity of transgenic Plasmodium falciparum expressing wild-type PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and dihydroartemisinin (C). The growth of parasites treated with pyrimethamine, chloroquine and dihydroartemisinin was detected using the SYBR Green I staining assay. The percentage of parasite growth was plotted against drug concentrations. Data were shown as mean ± S.D. of at least 3 independent experiments. K1: P. falciparum K1CB1; TM4: P. falciparum TM4/8.2; B2: transgenic PfPvDTclB2.

Mentions: Transgenic PfPvDTclB2 parasite was evaluated against standard anti-malarial drugs. Pyrimethamine is the standard antifolate drug and used as the primary compound to validate this system. As shown in Figure 3A and Table 1, the transgenic PfPvDTclB2 parasite was much more sensitive to pyrimethamine than the parental K1CB1 line, verifying that the wild-type Pvdhfr-ts gene replacing the Pfdhfr-ts gene is a pyrimethamine-sensitive variant. Moreover, the level of pyrimethamine sensitivity in the transgenic PfPvDTclB2 parasite is the same as the antifolate-sensitive P. falciparum TM4/8.2 strain (IC50 = 0.03 ± 0.02 µM). The parental K1CB1 line is also resistant to chloroquine, a 4-aminoquinoline drug that inhibits haemozoin formation in the food vacuole of the parasites. This transgenic PfPvDTclB2 line shows the same chloroquine-resistant phenotype as the parental P. falciparum K1CB1 strain, with IC50 values of 49.5 ± 5.8 nM and 46.0 ± 3.1 nM respectively (Figure 3B and Table 1), indicating that the dhfr-ts gene replacement did not affect sensitivity to drugs not targeting DHFR-TS. Another non-antifolate drug control used in this study was dihydroartemisinin (DHA). All parasites tested in this study were sensitive to DHA at the IC50 values of 0.6 ± 0.1 nM, 0.7 ± 0.3 nM and 0.4 ± 0.1 nM for P. falciparum TM4/8.2, K1CB1 and transgenic PfPvDTclB2, respectively (Figure 3C and Table 1).


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

Somsak V, Uthaipibull C, Prommana P, Srichairatanakool S, Yuthavong Y, Kamchonwongpaisan S - Malar. J. (2011)

Sensitivity of transgenic Plasmodium falciparum expressing wild-type PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and dihydroartemisinin (C). The growth of parasites treated with pyrimethamine, chloroquine and dihydroartemisinin was detected using the SYBR Green I staining assay. The percentage of parasite growth was plotted against drug concentrations. Data were shown as mean ± S.D. of at least 3 independent experiments. K1: P. falciparum K1CB1; TM4: P. falciparum TM4/8.2; B2: transgenic PfPvDTclB2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198988&req=5

Figure 3: Sensitivity of transgenic Plasmodium falciparum expressing wild-type PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and dihydroartemisinin (C). The growth of parasites treated with pyrimethamine, chloroquine and dihydroartemisinin was detected using the SYBR Green I staining assay. The percentage of parasite growth was plotted against drug concentrations. Data were shown as mean ± S.D. of at least 3 independent experiments. K1: P. falciparum K1CB1; TM4: P. falciparum TM4/8.2; B2: transgenic PfPvDTclB2.
Mentions: Transgenic PfPvDTclB2 parasite was evaluated against standard anti-malarial drugs. Pyrimethamine is the standard antifolate drug and used as the primary compound to validate this system. As shown in Figure 3A and Table 1, the transgenic PfPvDTclB2 parasite was much more sensitive to pyrimethamine than the parental K1CB1 line, verifying that the wild-type Pvdhfr-ts gene replacing the Pfdhfr-ts gene is a pyrimethamine-sensitive variant. Moreover, the level of pyrimethamine sensitivity in the transgenic PfPvDTclB2 parasite is the same as the antifolate-sensitive P. falciparum TM4/8.2 strain (IC50 = 0.03 ± 0.02 µM). The parental K1CB1 line is also resistant to chloroquine, a 4-aminoquinoline drug that inhibits haemozoin formation in the food vacuole of the parasites. This transgenic PfPvDTclB2 line shows the same chloroquine-resistant phenotype as the parental P. falciparum K1CB1 strain, with IC50 values of 49.5 ± 5.8 nM and 46.0 ± 3.1 nM respectively (Figure 3B and Table 1), indicating that the dhfr-ts gene replacement did not affect sensitivity to drugs not targeting DHFR-TS. Another non-antifolate drug control used in this study was dihydroartemisinin (DHA). All parasites tested in this study were sensitive to DHA at the IC50 values of 0.6 ± 0.1 nM, 0.7 ± 0.3 nM and 0.4 ± 0.1 nM for P. falciparum TM4/8.2, K1CB1 and transgenic PfPvDTclB2, respectively (Figure 3C and Table 1).

Bottom Line: 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.The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus