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J. Biol. Chem., Vol. 279, Issue 18, 18575-18582, April 30, 2004

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Inactivation of the Leishmania tarentolae Pterin Transporter (BT1) and Reductase (PTR1) Genes Leads to Viable Parasites with Changes in Folate Metabolism and Hypersensitivity to the Antifolate Methotrexate^*

Amal El Fadili, Christoph Kündig, Gaétan Roy, and Marc Ouellette

From the Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier de l'Université Laval (CHUL) and Division de Microbiologie, Faculté de Médecine, Université Laval, Québec G1V 4G2, Canada

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.

Received for publication, January 20, 2004 , and in revised form, February 18, 2004.

^* This work was supported in part by the Canadian Institute of Health Research (to M. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Holds a Canada Research Chair in Antimicrobial Resistance and a Burroughs Wellcome Fund Scholar in Molecular Parasitology. To whom correspondence should be addressed: Centre de Recherche en Infectiologie, CHUQ, pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2715; E-mail: marc.ouellette{at}crchul.ulaval.ca.

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