Complex Interplay between Sphingolipid and Sterol Metabolism Revealed by Perturbations to the Leishmania Metabolome Caused by Miltefosine

Abstract

With the World Health Organization reporting over 30,000 deaths and 200,000 to 400,000 new cases annually, visceral leishmaniasis is a serious disease affecting some of the world's poorest people. As drug resistance continues to rise, there is a huge unmet need to improve treatment. Miltefosine remains one of the main treatments for leishmaniasis, yet its mode of action (MoA) is still unknown. Understanding the MoA of this drug and parasite response to treatment could help pave the way for new and more successful treatments for leishmaniasis. A novel method has been devised to study the metabolome and lipidome of Leishmania donovani axenic amastigotes treated with miltefosine. Miltefosine caused a dramatic decrease in many membrane phospholipids (PLs), in addition to amino acid pools, while sphingolipids (SLs) and sterols increased. Leishmania major promastigotes devoid of SL biosynthesis through loss of the serine palmitoyl transferase gene (ΔLCB2) were 3-fold less sensitive to miltefosine than wild-type (WT) parasites. Changes in the metabolome and lipidome of miltefosine-treated L. major mirrored those of L. donovani A lack of SLs in the ΔLCB2 mutant was matched by substantial alterations in sterol content. Together, these data indicate that SLs and ergosterol are important for miltefosine sensitivity and, perhaps, MoA.

Keywords: Leishmania; lipid metabolism; mechanisms of action; metabolomics; miltefosine.

FIG 1

Effect of miltefosine on arginine metabolism observed in L. donovani axenic amastigotes after 24 h of exposure at the lower dose of 4.47 μM. Plots show peak area abundances detected in samples: untreated parasites are in blue, and parasites treated with 4.47 μM miltefosine are in red.

FIG 2

Effect of miltefosine on the SL biosynthetic pathway observed in L. donovani axenic amastigotes after 24 h of exposure at the lower dose of 4.47 μM. Plots show peak area abundances for key SLs detected in samples: untreated parasites are in blue, parasites treated with 4.47 μM miltefosine are in red. Sphinganine shown is the C₁₆ form, ceramide shown is the 34:1 form, sphingosine shown is the 18:1 form, and ceramide phosphate shown is 26:1 form. Plots show trends representative of all detected SLs of their type. For full list of detected SLs, refer to Table S3.

FIG 3

Fold changes in abundance of all SLs detected in L. donovani axenic amastigotes and/or L. major promastigotes, comparing treated parasites to untreated parasites. Lower-dose and higher-dose data are shown for each, corresponding to 4.47 μM and 13.41 μM, respectively, for L. donovani and 10 μM and 30 μM, respectively, for L. major. SLs that were not detected in a certain data set are marked with ND, while X denotes complete absence in drug-treated parasites and presence in untreated parasites.

FIG 4

Ergosterol biosynthesis pathway. Sterols that increased, decreased, or were detected but with no change as a response to miltefosine exposure are shown on the pathway (L. donovani axenic amastigotes with 5-h drug exposure in blue, L. donovani axenic amastigotes with 24-h drug exposure in red, L. major wild-type promastigotes in green). In all cases, trends were seen for both low and high concentrations of miltefosine treatment. Chromatographic peaks shown for ergosterol, cholesterol, 5,7,24(28)-ergostatrienol, and 5-dehydroepisterol in L. major wild type (black trace) and ΔLCB2 mutants (red trace) to highlight the differences in sterol profiles between them. 5,7,24(28)-Ergostatrienol and 5-dehydroepisterol share the same logP, and therefore, it is not possible to distinguish to which peak (9.8 to 9.9 min or 10.2 to 10.3 min) these sterols correspond.