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Antimicrobial Agents and Chemotherapy, July 2002, p. 2145-2154, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2145-2154.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Recombinant Pneumocystis carinii Topoisomerase I: Differential Interactions with Human Topoisomerase I Poisons and Pentamidine

Rukiyah T. Van Dross,{dagger} and Marilyn M. Sanders*

Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

Received 23 January 2002/ Returned for modification 7 March 2002/ Accepted 10 April 2002

The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The P. carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg2+ requirement. Cleavage assays reveal that both the human and P. carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4',6'-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that P. carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme, P. carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of P. carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with P. carinii topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.

* Corresponding author. Mailing address: Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-4593. Fax: (732) 235-4073. E-mail: msanders{at}umdnj.edu.

{dagger} Present address: Department of Pathology, University of Kansas Medical Center, Kansas City, KS 66160.

Antimicrobial Agents and Chemotherapy, July 2002, p. 2145-2154, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2145-2154.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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