Metabolic adaptations of Leishmania donovani in relation to differentiation, drug resistance, and drug pressure

Abstract

SummaryAntimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG-resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC-MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSGsusceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG-resistant and SSG-sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG-resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG-resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG-resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under Sb III drug pressure.

Experimental Resistance to Drug Combinations in Leishmania donovani: Metabolic and Phenotypic Adaptations

“…Membrane fluidity was tested with a fluorescence anisotropy assay and showed that all membranes from the resistant lines (except M) present lower fluidity. Similar results (lower fluidity) have been obtained and suggested in the resistance of Leishmania parasites to tafenoquine (42) and MIL (65) but are different from the results obtained with resistance to PMM (24), antimonials (7,66), and AmB (12), for which membrane fluidity was found to be higher. Our results are supported by the fact that an increase in six low unsaturated GPLs and a decrease in six high unsaturated GPLs were detected in the A line, which was expected to result in a decrease in membrane fluidity.…”

“…All spectra were collected in continuous single mass spectrometry (MS) mode. A sample list setup was performed as described previously (7,31), and data processing and metabolite identification were performed in mzMatch, as described extensively in Berg et al (7). Briefly, the selected mass chromatograms were putatively identified by matching the masses progressively to those from metabolite-specific databases (mass accuracy, Ͻ2 ppm, after correction for loss or gain of a proton in negative-mode or positive-mode ESI, respectively).…”

“…Antimonials (Sb) have a complex (and not yet fully understood) multifactorial mode of action involving (i) a direct inhibitory effect of Sb III on trypanothione reductase of the parasite (4) and (ii) an indirect effect of Sb V by the stimulation of macrophages that kill their intracellular invaders (5). Several molecular adaptations have been encountered in Sb-resistant clinical isolates (including genomic structural variation and a wide range of metabolic changes in RNA-DNA synthesis, phospholipid, and energy metabolism), suggesting a complex multifactorial mechanism of resistance (6,7). Amphotericin B (AmB) or one of its lipid-carrier formulations is another highly effective antileishmanial agent often used in second-line treatments (8,9).…”

Genomic Appraisal of the Multifactorial Basis for In Vitro Acquisition of Miltefosine Resistance in Leishmania donovani

“…Rakotomanga et al (39) were able to correlate decreased fluidity with reduced HePC-external phospholipid monolayer interactions. Interestingly, changes in membrane fluidity in Leishmania have been ascribed to antimonial (40) or amphotericin B (41) resistance. The BSA used in our experiments to remove surfaceadsorbed HePC-BODIPY did not remove HePC inserted in the internal membrane leaflet; thus, our uptake fraction may have included both inserted and incorporated HePC-BODIPY.…”

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…74 As a result of this high incidence of resistance, there are several studies looking at the metabolic differences in clinical resistant isolates. For example, sodium stibogluconate-resistant promastigote isolates were compared with sensitive isolates from the Indian subcontinent, 30 in the presence and absence of drug pressure and during the parasite cell cycle using untargeted Orbitrap mass spectrometry coupled to a zic-HILIC column. 30 The MOA of antimony proved elusive using this technique, as large areas of metabolism were affected.…”