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.
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.
Pourshafie
Mohammad
M
UMR 8076 CNRS, Faculté de Pharmacie, Université Paris XI, 5 rue Jean-Baptiste Clément, 92290-Châtenay-Malabry, France.
Morand
Stanislas
S
Virion
Alain
A
Rakotomanga
Michaelle
M
Dupuy
Corinne
C
Loiseau
Philippe M
PM
D000478:Alkylation; D000595:Amino Acid Sequence; D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004351:Drug Resistance; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification; D016053:RNA, Protozoan / Q000032:analysis* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D013261:Sterols / Q000032:analysis / Q000378:metabolism
D000981:Antiprotozoal Agents; D012333:RNA, Messenger; D016053:RNA, Protozoan; D013261:Sterols; D000666:Amphotericin B; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase