Mechanism of amphotericin B resistance in Leishmania donovani promastigotes

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-3beta-ol and not ergosterol as in the AmB-sensitive strain.

FIG. 1

Accumulation of AmB in L. donovani DD8-sensitive (S) and -resistant (R) promastigotes after incubation with 0.2 μM AmB. Values are means of three independent experiments, and standard deviations are represented by error bars.

FIG. 2

Accumulation of AmB in L. donovani DD8-sensitive (A) and -resistant (R) promastigotes treated for 2 h. Values are means of three independent experiments, and standard deviations are represented by error bars.

FIG. 3

Efflux of AmB from treated L. donovani DD8-sensitive (S) and -resistant (R) promastigotes for 2 h at 0.2 μM. Error bars indicate standard deviation.

FIG. 4

Proposed biosynthetic pathway of ergosterol in L. donovani. Symbols: →, pathway of wild-type promastigotes; ⇒, pathway of AmB-resistant promastigotes; ▪, sites inhibited by ketoconazole; ……→, defective 24-methyltransferase in AmB-resistant promastigotes.