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Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.

ABSTRACT: Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.

SUBMITTER: Tirakarn S

PROVIDER: S-EPMC3444756 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.

Tirakarn Srisuda S Riangrungroj Pinpunya P Kongsaeree Palangpon P Imwong Mallika M Yuthavong Yongyuth Y Leartsakulpanich Ubolsree U

Parasitology international 20120102 2

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is sim ...[more]

PMID: 22234170

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Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae.

Project description:Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).

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Functional analysis of Plasmodium vivax dihydrofolate reductase-thymidylate synthase genes through stable transformation of Plasmodium falciparum.

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