Affiliation:
1. Chimiothérapie Antiparasitaire, UMR 8076 CNRS
2. INSERM U486, IFR 75-ISIT Faculté de Pharmacie, Université Paris XI, 92290-ChÂtenay-Malabry, France
Abstract
ABSTRACT
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-Δ-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
.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Reference22 articles.
1. Carrier effects on biological activity of amphotericin B
2. Amphotericin B: current understanding of mechanisms of action
3. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.162:156-159.
4. Croft, S. L., and V. Yardley. 2002. Chemotherapy of leishmaniasis. Curr. Pharm. Des.8:319-342.
5. Desjeux, P., B. Piot, K. O'Neil, and J. P. Meert. 2001. Co-infections of Leishmania/HIV in south Europe. Med. Trop.61:187-193.
Cited by
63 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献