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Antimicrobial Agents and Chemotherapy, April 2001, p. 1271-1277, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1271-1277.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

In Vitro Generation of Novel Pyrimethamine Resistance Mutations in the Toxoplasma gondii Dihydrofolate Reductase

Mary G. Reynolds,dagger Jung Oh,Dagger and David S. Roos*

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received 13 July 2000/Returned for modification 28 August 2000/Accepted 3 January 2001

Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-ts protein sequences. To assess the potential for pyrimethamine-resistant dhfr mutants to emerge, three drug-sensitive variants of the T. gondii dhfr-ts gene (the wild-type T. gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T. gondii parasites or dhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trpright-arrowArg), 98 (Leuright-arrowSer), and 134 (Leuright-arrowHis).

* Corresponding author. Mailing address: Department of Biology, University of Pennsylvania, 415 South University Ave., Philadelphia, PA 19104-6018. Phone: (215) 898-2118. Fax: (215) 898-8780. E-mail: droos{at}sas.upenn.edu.

dagger Present address: Department of Biology, Emory University, Atlanta, GA 30322.

Dagger Present address: Washington University School of Medicine, St. Louis, MO 63110.

Antimicrobial Agents and Chemotherapy, April 2001, p. 1271-1277, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1271-1277.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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