Abstract
Leishmania infantum is an etiological agent of the life-threatening visceral form of leishmaniasis. Liposomal amphotericin B (AmB) followed by a short administration of miltefosine (MF) is a drug combination effective for treating visceral leishmaniasis in endemic regions of India. Resistance to MF can be due to point mutations in the miltefosine transporter (MT). Here we show that mutations in MT are also observed in Leishmania AmB-resistant mutants. The MF-induced MT mutations, but not the AmB induced mutations in MT, alter the translocation/uptake of MF. Moreover, mutations in the MT selected by AmB or MF have a major impact on lipid species that is linked to cross-resistance between both drugs. These alterations include changes of specific phospholipids, some of which are enriched with cyclopropanated fatty acids, as well as an increase in inositolphosphoceramide species. Collectively these results provide evidence of the risk of cross-resistance emergence derived from current AmB-MF sequential or co-treatments for visceral leishmaniasis.
Highlights
Protozoan parasites belonging to the Leishmania genus cause several vector-borne diseases collectively referred as leishmaniases
We demonstrate here that different point mutations in a Ptype ATPase could play an important role in resistance, to miltefosine, and to amphotericin B
The Leishmania infantum (MHOM/MA/67/ITMAP-263) wild-type strain (Ldi263 wt) and the in vitro generated resistant mutants AmB1000.1 and MF200.5 [23, 24], which are respectively resistant to 1000 nM of AmB and 200 μM MF, were grown in SDM-79 medium at 25 ̊C supplemented with 10% fetal bovine serum, 5 μg/mL of haemin at pH 7.0 with either 200 μM of MF (Miltefosine, Cayman Chem.) or 1 μM AmB (Amphotericin B solution, Sigma) for the mutant strains, and 40 μg/mL G418 (Geneticin, Gibco-BRL) for the episomal overexpressors
Summary
Protozoan parasites belonging to the Leishmania genus cause several vector-borne diseases collectively referred as leishmaniases. The old-fashioned and toxic antimonial derivatives top the short list of registered compounds against Leishmania spp. In addition to their toxicity, pentavalent antimonials require long treatment schedules and are associated with resistance [1, 3]. Clinical resistance to AmB is rare [5] but a recent study in India has reported a L. donovani field strain resistant to AmB [6]. Another leishmanicidal drug introduced in the early 21st century is the alkyl-phospholipid analogue miltefosine (MF). Since its registration in 2002, it has had increasing relapse rates and the emergence of drug resistance strains [7, 8]
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