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Antimicrobial Agents and Chemotherapy, November 2008, p. 4072-4080, Vol. 52, No. 11
0066-4804/08/$08.00+0 doi:10.1128/AAC.00384-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Cloning, Expression, and Characterization of Babesia gibsoni Dihydrofolate Reductase-Thymidylate Synthase: Inhibitory Effect of Antifolates on Its Catalytic Activity and Parasite Proliferation
Gabriel O. Aboge,1
Honglin Jia,1
Mohamad A. Terkawi,1
Youn-Kyoung Goo,1
Yoshifumi Nishikawa,1
Fujiko Sunaga,2
Kuzuhiko Namikawa,2
Naotoshi Tsuji,3
Ikuo Igarashi,1
Hiroshi Suzuki,1
Kozo Fujisaki,1,4 and
Xuenan Xuan1*
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan,1 Department of Infectious Diseases, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229-8501, Japan,2 National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan,3 Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan4
Received 20 March 2008/ Returned for modification 18 July 2008/ Accepted 30 August 2008
Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed Km values of 4.70 ± 0.059 (mean ± standard error of the mean) and 9.75 ± 1.64 µM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC50] = 68.6 ± 5.20 nM) than pyrimethamine (IC50 = 55.0 ± 2.08 µM) and trimethoprim (IC50 = 50 ± 12.5 µM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.
* Corresponding author. Mailing address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5648. Fax: 81-155-49-5643. E-mail: gen{at}obihiro.ac.jp
Published ahead of print on 15 September 2008.
Antimicrobial Agents and Chemotherapy, November 2008, p. 4072-4080, Vol. 52, No. 11
0066-4804/08/$08.00+0 doi:10.1128/AAC.00384-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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