EFFECT OF KETOCONAZOLE ON LETHAL ACTION OF AMPHOTERICIN-B ON LEISHMANIA-MEXICANA PROMASTIGOTES

The effect of ergosterol depletion by ketocouazole on the leishmanicidal activity of the pore-forming antibiotic amphotericin B (AmB) was investigated. Leishmania mexicana promastigotes were lysed within minutes by the addition of micromolar concentrations of AmB (0.5 mu M) but became insensitive to AmB after growth in the presence of ketoconazole (0.25 mu M, 90 h). Lipid chromatographic analysis indicated that under such conditions, ketoconazole depleted the major Leishmania sterols, dehydroepisterol and ergosterol. Plasma membrane vesicles prepared from ketoconazole-treated promastigotes exhibited a much reduced enhancement of their salt permeability after the addition of AmB at concentrations as high as 5 mu M. This finding clearly indicates that upon ketoconazole treatment, the capacity of pore formation by the antibiotic is substantially impaired. The reduction of desmethyl sterols by ketoconazole was accompanied by a significant increase of 14-alpha-methyl sterols, but exogenous cholesterol remained unchanged. This ability of leishmania promastigotes to incorporate cholesterol from the external medium may explain why ketoconazole-treated cells exhibited a much decreased but significative response to AmB when they were exposed to high AmB concentrations (2.5 or 5.0 mu M). Parallel measurements by using a fluorescence energy transfer method indicated that binding of AmB to ketoconazole-treated Leishmania promastigotes and heat-transformed leishmanias was also decreased but to different extents, a finding that may be related to the differences in their sterol content. The results obtained clearly indicate that the specific interaction of AmB with desmethyl sterols, such as dehydroepisterol, ergosterol, and even exogenous cholesterol, is an absolute requirement for the lethal action exerted by this polyene antibiotic on L. mexicana promastigotes.