Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes.
OBJECTIVE
This study focuses on the importance of sterols in the action of miltefosine (hexadecylphosphocholine, HePC) against Leishmania donovani.


METHODS
Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-beta-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC-lipid interactions.


RESULTS
CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased.


CONCLUSIONS
Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, (1)H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.
Saint-Pierre-Chazalet
M
M
Université Paris-Sud, UMR 8076, Chimiothérapie Antiparasitaire, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, Chatenay-Malabry, France.
Ben Brahim
M
M
Le Moyec
L
L
Bories
C
C
Rakotomanga
M
M
Loiseau
P M
PM
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry*; D004351:Drug Resistance; D007893:Leishmania donovani / Q000187:drug effects*; D050356:Lipid Metabolism / Q000187:drug effects; D050296:Microbial Viability; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D021261:Parasitic Sensitivity Tests; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013261:Sterols / Q000032:analysis*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D000981:Antiprotozoal Agents; D013261:Sterols; D013778:Tetrazolium Salts; D013844:Thiazoles; D010767:Phosphorylcholine; C039128:miltefosine; C022616:thiazolyl blue