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Antimicrobial Agents and Chemotherapy, July 2004, p. 2409-2414, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2409-2414.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning of S-Adenosyl-L-Methionine:C-24-{Delta}-Sterol-Methyltransferase (ERG6) from Leishmania donovani and Characterization of mRNAs in Wild-Type and Amphotericin B-Resistant Promastigotes

Mohammad Pourshafie,1 Stanislas Morand,2 Alain Virion,2 Michaelle Rakotomanga,1 Corinne Dupuy,2 and Philippe M. Loiseau1*

Chimiothérapie Antiparasitaire, UMR 8076 CNRS,1 INSERM U486, IFR 75-ISIT Faculté de Pharmacie, Université Paris XI, 92290-ChÂtenay-Malabry, France2

Received 2 December 2003/ Returned for modification 2 February 2004/ Accepted 17 March 2004

The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant Leishmania donovani promastigotes, suggesting that the S- adenosyl-L-methionine:C-24-{Delta}-sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites. From an L. donovani wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position. The two cDNAs differed by their 3'-untranslated region (3'-UTR) and 5'-UTR sequences. One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells. The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells. As a functional test, ERG6 null mutant Saccharomyces cerevisiae yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate SCMT1/ERG6 coding sequence cloned from L. donovani. The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation. In AmB-R L. donovani promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells. Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in Leishmania.

* Corresponding author. Mailing address: UMR 8076 CNRS, Faculté de Pharmacie, Université Paris XI, 5 rue Jean-Baptiste Clément, 92290-ChÂtenay-Malabry, France. Phone: (33) 1 46 83 55 53. Fax: (33) 1 46 83 55 57. e-mail: Philippe.Loiseau{at}cep.u-psud.fr.

Antimicrobial Agents and Chemotherapy, July 2004, p. 2409-2414, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2409-2414.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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