Antimicrob Agents Chemother 42(2): 352-357

Mechanism of Amphotericin B Resistance in <em>Leishmania donovani</em> Promastigotes

Institut de Chimie des Substances Naturelles, CNRS, F-91190, Gif sur Yvette,^{and Biologie et Contrôle des Organismes Parasites, Faculté de Pharmacie, Université de Paris-Sud, F-92296, Châtenay-Malabry,}^France

^{Corresponding author. Mailing address: Biologie et Contrôle des Organismes Parasites, Faculté de Pharmacie, Université de Paris-Sud, F-92296, Châtenay-Malabry, France. Phone: 33 1 46 83 55 54. Fax: 33 1 46 83 55 57. E-mail:}rf.dusp-u.pec@uaesiol.eppilihp.

Received 1997 Jun 23; Revisions requested 1997 Nov 17; Accepted 1997 Dec 3.

Abstract

Amphotericin B (AmB)-resistant Leishmania donovani promastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3β-ol and not ergosterol as in the AmB-sensitive strain.

Abstract

Leishmania donovani, a protozoan parasite, is the causative agent of visceral leishmaniasis, a disease which is fatal in the absence of treatment. Its incidence is about 0.5 million cases annually (28). Pentavalent antimonials (Pentostam and Glucantime) are the first-line drugs, but refractory strains to these drugs are increasingly prevalent in areas of endemicity (26, 33). Amphotericin B (AmB) is a polyene antibiotic which binds preferentially to ergosterol, the major sterol of fungi, Leishmania, and Trypanosoma cruzi. This antibiotic is a second-line treatment for leishmaniasis (1), but side effects limit its use. Recently, AmB-lipid formulations (AmBisome and Amphocil) have been developed with reduced toxicity and an improved therapeutic index. They represent an alternative for the treatment of visceral leishmaniasis (6, 23, 31). As AmB formulations are increasingly being used for the treatment of visceral leishmaniasis and probably mucocutaneous leishmaniasis, we decided to investigate the development and mechanism of resistance. Resistance to AmB is uncommon in fungi but has been reported previously (16). The aim of our work was to establish a line of AmB-resistant L. donovani promastigotes in vitro and to study the molecular basis of this resistance.

ACKNOWLEDGMENT

We thank S. L. Croft for critical reading of the manuscript.

ACKNOWLEDGMENT

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