Mechanisms of experimental resistance of Leishmania to miltefosine: Implications for clinical use

Chemical structure of MIL.

Binding and uptake of MIL in Leishmania parasites. Sequential steps in the accumulation of MIL: (A) Binding of the drug to the outer leaflet of the plasma membrane. MIL is recruited by bovine serum albumin (BSA), which acts as a reservoir for the drug. (B) The fraction bound to cell membranes is internalized through a flippase protein (F) machinery present at the lipid bilayer. This translocation machinery includes, at least, two proteins at the plasma membrane: the MIL transporter LdMT and its beta subunit LdRos3 (see in the text).

LdMT and LdCDC50 are normally localized at the parasite plasma membrane. LdMT−/− parasites (lacking LdMT protein) (A and C) and M-1M parasites (lacking LdRos3) (B and D) were transfected with LdMT-GFP (A and B) or LdRos3-GFP (C and D) and the localization of the GFP chimeras were studied by fluorescence images of hypotonic buffer-treated parasites. LdMT and LdCDC50 are localized at the plasma membrane (A and D), but in the absence of the corresponding subunits (B, C) are localized in intracellular organelles (Bar, 5μm). Adapted from Pérez-Victoria et al. (manuscript in preparation).

Dependence of both LdMT and LdRos3 for the uptake of [¹⁴C]MLF and correlation between their expression and uptake levels in L. donovani. The uptake of [¹⁴C]MLF was measured after 60min incubations for the following lines: wild-type parasites (Wt); M-40 R parasites (M-40 R), which contain inactivating mutations in LdMT; M-40 R parasites transfected with LdRos3 (β); M-40 R transfected with the pXG′-LdMT-GFP construction and selected against increasing concentrations of G-418 (15, 50, 100 and 500μg/ml); M-1M parasites (M-1M), which contains inactive LdRos3 alleles; M-1M parasites transfected with LdMT (M-1M LdMT); M-1M parasites double transfected, firstly with LdMT and selected with 100μg/ml of hygromycin B and later with LdRos3-GFP (M-1M+LdMT+LdRos3-GFP) selected with increasing concentrations of G-418 (10, 20, 50 and 100μg/ml); wild-type parasites transfected with either LdRos3 (Wt β) or LdMT (Wt α). Bars shown are the mean±S.D. of three independent experiments. Correlation between uptake and expression levels is further highlighted by western blots with anti-GFP antibodies, shown above the bars. Adapted from Pérez-Victoria et al. (2003b, manuscript in preparation).