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Antimicrobial Agents and Chemotherapy, April 2001, p. 1168-1173, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1168-1173.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

DNA Transformation of Leishmania infantum Axenic Amastigotes and Their Use in Drug Screening

Denis Sereno,1,dagger Gaétan Roy,1 Jean Loup Lemesre,2 Barbara Papadopoulou,1 and Marc Ouellette1,*

Centre de Recherche en Infectiologie du Centre de Recherche du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada,1 and Laboratoire de Biologie et Immunologie Parasitaire, Centre IRD de Montpellier, Montpellier, France2

Received 18 September 2000/Returned for modification 20 November 2000/Accepted 24 January 2001

Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 µM (0.12 µg/ml), 55 µg/ml, 95 µg/ml, and 0.12 µg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie, CHUQ, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec, Canada G1V 4G2. Phone: 418-654 2705. Fax: 418-654 2715. E-mail: Marc.Ouellette{at}crchul.ulaval.ca.

dagger Present address: Centre IRD de Montpellier, Montpellier, France.

Antimicrobial Agents and Chemotherapy, April 2001, p. 1168-1173, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1168-1173.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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