The rapid emergence of metabolomics has enabled system-wide measurements of metabolites in various organisms. However, advances in the mechanistic understanding of metabolic networks remain limited...

Metabolic pathways in Magnetospirillum gryphiswaldense MSR-1 are significantly altered under microaerobic (O ₂ -limited) growth conditions enabling magnetosome formation.

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate. In this work we have followed the distribution of carbon derived from glucose in bloodstream form trypanosomes, the causative agent of African trypanosomiasis, revealing it to enter a diverse range of metabolites. The work involved using ¹³C-labelled glucose and following the fate of the labelled carbon with an LC-MS based metabolomics platform. Beyond glycolysis and the oxidative branch of the pentose phosphate pathway the label entered lipid biosynthesis both through glycerol 3-phosphate and also acetate. Glucose derived carbon also entered nucleotide synthesis through ribose and pyrimidine synthesis through oxaloacetate-derived aspartate. Appreciable quantities of the carboxylic acids succinate and malate were identified, although labelling patterns indicate they are not TCA cycle derived. Amino sugars and sugar nucleotides were also labelled as was inositol used in protein modification but not in inositol phospholipid headgroup production. We confirm active and essential oxaloacetate production in bloodstream form trypanosomes and show that phosphoenolpyruvate carboxykinase is essential to these parasites using RNA interference. The amount of glucose entering these metabolites is minor compared to the quantity that enters pyruvate excreted from the cell, but the observation that enzymes contributing to the metabolism of glucose beyond glycolysis can be essential offers potential new targets for chemotherapy against trypanosomiasis.

In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei . The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.

Introduction: Macrophages constitute a heterogeneous population of functionally distinct cells involved in several physiological and pathological processes. They display remarkable plasticity by changing their phenotype and function in response to environmental cues representing a spectrum of different functional phenotypes. The so-called M1 and M2 macrophages are often considered as representative of pro- and anti-inflammatory ends of such spectrum. Metabolomics approach is a powerful tool providing important chemical information about the cellular phenotype of living systems, and the changes in their metabolic pathways in response to various perturbations.Objectives: This study aimed to characterise M1 and M2 phenotypes in THP-1 macrophages in order to identify characteristic metabolites of each polarisation state.Methods: Herein, untargeted liquid chromatography (LC)–mass spectrometry (MS)-based metabolite profiling was applied to characterise the metabolic profile of M1-like and M2-like THP-1 macrophages.Results: The results showed that M1 and M2 macrophages have distinct metabolic profiles. Sphingolipid and pyrimidine metabolism was significantly changed in M1 macrophages whereas arginine, proline, alanine, aspartate and glutamate metabolism was significantly altered in M2 macrophages.Conclusion: This study represents successful application of LC–MS metabolomics approach to characterise M1 and M2 macrophages providing functional readouts that show unique metabolic signature for each phenotype. These data could contribute to a better understanding of M1 and M2 functional properties and could pave the way for developing new therapeutics targeting different immune diseases.

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself. Antimicrobial resistance threatens to reverse many of the great strides made against pathogens responsible for disease. Understanding the molecular processes underlying resistance is crucial to quantifying and tackling the problem. Here we select resistance in Leishmania parasites to amphotericin B, an antileishmanial drug of increasing importance. We then combine genome sequencing with untargeted and targeted metabolomics analyses to identify a gene, sterol 14α-demethylase, mutation of which drives a change in sterol metabolism and loss of ergosterol, the molecular target of amphotericin B. Accumulation of a downstream intermediate of ergosterol biosynthesis indicated the enzyme itself retains activity, but the pathway to ergosterol is truncated. Expression of wild-type sterol 14α-demethylase in the resistant cells restored amphotericin B sensitivity and normal ergosterol production.