Untargeted metabolomics to understand the basis of phenotypic differences in amphotericin B-resistant Leishmania parasites.

Andrew W Pountain,Michael P Barrett

doi:10.12688/wellcomeopenres.15452.1

Abstract

Background: Protozoan Leishmania parasites are responsible for a range of clinical infections that represent a substantial challenge for global health. Amphotericin B (AmB) is increasingly used to treat Leishmania infection, so understanding the potential for resistance to this drug is an important priority. Previously we described four independently-derived AmB-resistant L. mexicana lines that exhibited resistance-associated genetic lesions resulting in altered sterol content. However, substantial phenotypic variation between these lines, including differences in virulence attributes, were not fully explained by these changes. Methods: To identify alterations in cellular metabolism potentially related to phenotypic differences between wild-type and AmB-resistant lines, we extracted metabolites and performed untargeted metabolomics by liquid chromatography-mass spectrometry. Results: We observed substantial differences in metabolite abundance between lines, arising in an apparently stochastic manner. Concerted remodeling of central carbon metabolism was not observed; however, in three lines, decreased abundance of several oligohexoses was observed. Given that the oligomannose mannogen is an important virulence factor in Leishmania, this could relate to loss of virulence in these lines. Increased abundance of the reduced forms of the oxidative stress-protective thiols trypanothione and glutathione was also observed in multiple lines. Conclusions: This dataset will provide a useful resource for understanding the molecular basis of drug resistance in Leishmania, and suggests a role for metabolic changes separate from the primary mechanism of drug resistance in determining the phenotypic profile of parasite lines subjected to experimental selection of resistance.

Highlights

Parasites of the genus Leishmania place a high burden on public health, with clinical manifestations ranging from self-healing cutaneous lesions to life-threatening visceral leishmaniasis
Loss of miltefosine transporter function was previously reported in an AmBresistant line[13], and an RNAi knockdown screen in the related parasite Trypanosoma brucei revealed that reduced expression of the T. brucei ortholog of LmxM.13.1530 is associated with Amphotericin B (AmB) resistance[18]
Changes in carbohydrate and energy metabolism Since overexpression of PPP enzymes leads to greater tolerance of oxidative stress and AmB resistance[20], we investigated whether AmBR lines had a higher abundance of PPP intermediates

Summary

Introduction

Parasites of the genus Leishmania place a high burden on public health, with clinical manifestations ranging from self-healing cutaneous lesions to life-threatening visceral leishmaniasis Efforts to control this disease rely heavily on chemotherapeutic agents, but the traditional front-line class, pentavalent antimonials, suffers from both high toxicity and increasing drug resistance[1]. The mechanism of action of AmB is not fully understood but involves specific binding to ergosterol within parasite and fungal membranes, leading to disruption of membrane function either through the formation of aqueous pores or by sequestering ergosterol within the membrane[5,6] Resistance to this compound is not yet widespread in Leishmania populations but has been documented both in clinical isolates[7,8,9] and through experimental selection or genetic manipulation[10,11,12,13,14,15,16,17]. Conclusions: This dataset will provide a useful resource for understanding the molecular basis of drug resistance in Leishmania, and suggests a role for metabolic changes separate from the primary mechanism of drug resistance in determining the phenotypic profile of parasite lines subjected to experimental selection of resistance

Methods

Results

Conclusion