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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 berghei expressing PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and artesunate (C). The transgenic parasites were validated using the standard four-day suppressive test. Percent parasite inhibition was plotted for groups of at least five mice orally given varying doses of pyrimethamine, chloroquine and artesunate. Data were shown as mean ± S.D. of at least three independent experiments.
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Figure 4: Sensitivity of transgenic Plasmodium berghei expressing PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and artesunate (C). The transgenic parasites were validated using the standard four-day suppressive test. Percent parasite inhibition was plotted for groups of at least five mice orally given varying doses of pyrimethamine, chloroquine and artesunate. Data were shown as mean ± S.D. of at least three independent experiments.

Mentions: After inoculation, mice in the untreated control group showed a progressively increasing parasitaemia, and all the mice died by day 11 (data not shown). As shown in Figure 4A and Table 1, the transgenic PbPvDTcl4 demonstrated a drug susceptibility profile similar to that of the wild-type parental PbGFP with an ED50 of 0.53 ± 0.24 mg/kg and 0.69 ± 0.21 mg/kg, respectively. This demonstrated that the wild-type PvDHFR-TS enzyme was equally susceptible to the antifolate compound compared with wild-type PbDHFR-TS. In contrast, transgenic PbPvSP21cl2 was approximately 40-fold more resistant to pyrimethamine than the PbPvDTcl4 parasite line (Figure 4A). Therefore, the double mutant P. vivax DHFR-TS confers a high level of resistance to pyrimethamine in P. berghei.


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 berghei expressing PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and artesunate (C). The transgenic parasites were validated using the standard four-day suppressive test. Percent parasite inhibition was plotted for groups of at least five mice orally given varying doses of pyrimethamine, chloroquine and artesunate. Data were shown as mean ± S.D. of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sensitivity of transgenic Plasmodium berghei expressing PvDHFR-TS enzyme to pyrimethamine (A), chloroquine (B) and artesunate (C). The transgenic parasites were validated using the standard four-day suppressive test. Percent parasite inhibition was plotted for groups of at least five mice orally given varying doses of pyrimethamine, chloroquine and artesunate. Data were shown as mean ± S.D. of at least three independent experiments.
Mentions: After inoculation, mice in the untreated control group showed a progressively increasing parasitaemia, and all the mice died by day 11 (data not shown). As shown in Figure 4A and Table 1, the transgenic PbPvDTcl4 demonstrated a drug susceptibility profile similar to that of the wild-type parental PbGFP with an ED50 of 0.53 ± 0.24 mg/kg and 0.69 ± 0.21 mg/kg, respectively. This demonstrated that the wild-type PvDHFR-TS enzyme was equally susceptible to the antifolate compound compared with wild-type PbDHFR-TS. In contrast, transgenic PbPvSP21cl2 was approximately 40-fold more resistant to pyrimethamine than the PbPvDTcl4 parasite line (Figure 4A). Therefore, the double mutant P. vivax DHFR-TS confers a high level of resistance to pyrimethamine in P. berghei.

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