Metabolomics to Unveil and Understand Phenotypic Diversity between Pathogen Populations

Abstract

Leishmaniasis is a debilitating disease caused by the parasite Leishmania. There is extensive clinical polymorphism, including variable responsiveness to treatment. We study Leishmania donovani parasites isolated from visceral leishmaniasis patients in Nepal that responded differently to antimonial treatment due to differing intrinsic drug sensitivity of the parasites. Here, we present a proof-of-principle study in which we applied a metabolomics pipeline specifically developed for L. donovani to characterize the global metabolic differences between antimonial-sensitive and antimonial-resistant L. donovani isolates. Clones of drug-sensitive and drug-resistant parasite isolates from clinical samples were cultured in vitro and harvested for metabolomics analysis. The relative abundance of 340 metabolites was determined by ZIC-HILIC chromatography coupled to LTQ-Orbitrap mass spectrometry. Our measurements cover approximately 20% of the predicted core metabolome of Leishmania and additionally detected a large number of lipids. Drug-sensitive and drug-resistant parasites showed distinct metabolic profiles, and unsupervised clustering and principal component analysis clearly distinguished the two phenotypes. For 100 metabolites, the detected intensity differed more than three-fold between the 2 phenotypes. Many of these were in specific areas of lipid metabolism, suggesting that the membrane composition of the drug-resistant parasites is extensively modified. Untargeted metabolomics has been applied on clinical Leishmania isolates to uncover major metabolic differences between drug-sensitive and drug-resistant isolates. The identified major differences provide novel insights into the mechanisms involved in resistance to antimonial drugs, and facilitate investigations using targeted approaches to unravel the key changes mediating drug resistance.

Elucidation of Cellular Mechanisms Involved in Experimental Paromomycin Resistance in Leishmania donovani

“…Resistance to drugs often results in modifications of the membrane structure and lipid composition, as observed in SAG-resistant and amphotericin B-resistant L. donovani (19,26,29) Here we observed a significant increase in membrane fluidity in paromomycin-resistant L. donovani, indicating alteration of the membrane composition of the parasites. Further, the resistant parasites showed marked decreases in intracellular drug accumulation, in line with the previous findings that indicated the association of PMM resistance with decreased drug accumulation (14,16).…”

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

“…], 5 m; Merck/ SeQuant) and Zic-HILIC high-performance liquid chromatography (HPLC) column (150 mm by 2.1 mm i.d., 3.5 m, 100 Å; Merck/ SeQuant) was carried out with a gradient of (A) 0.1% formic acid in acetonitrile and (B) 0.1% formic acid in water. The flow rate was 100 l/min, with an injection volume of 10 l. Gradient elution was performed as described in t'Kindt et al (30). High-resolution mass measurements were obtained with an Exactive Orbitrap mass spectrometer (Thermo Fisher) at the Scottish Metabolomics Facility (Glasgow Polyomics, University of Glasgow, Glasgow, Scotland).…”

“…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). In a first round of identification, an in-house Leishmania database was used (based on LeishCyc and further completed with identifications from Lipid Metabolites and Pathways Strategy [MAPS] [30,32]). The remaining unidentified peaks were subjected to a second round of matching against the complete Lipid MAPS (32) and then against KEGG (33).…”

“…The different lines were grown synchronously, while the growth was monitored daily by microscopic counting; the different lines were all harvested in the stationary-growth stage for metabolite extraction. The metabolite extraction protocol was done as previously described (30).…”

Leishmania antimony resistance/ susceptibility in Algerian foci

“…Interestingly, a hierarchical clustering approach (antimony resistant and susceptible) revealed differences in the metabolite abundance for the drug-resistant and -sensitive clones [92]. However, in this study the sample size was not large enough to conclusively identify drug-resistant parasites.…”