On-line targeted metabolomics for real-time monitoring of relevant compounds in fermentation processes.

Fermentation monitoring is a powerful tool for bioprocess development and optimization. On-line metabolomics is a technology that is starting to gain attention as a bioprocess monitoring tool, allowing the direct measurement of many compounds in the fermentation broth at a very high time resolution. In this work, targeted on-line metabolomics was used to monitor 40 metabolites of interest during three Escherichia coli succinate production fermentation experiments every 5 min with a triple quadrupole mass spectrometer. This allowed capturing high-time resolution biological data that can provide critical information for process optimization. For nine of these metabolites, simple univariate regression models were used to model compound concentration from their on-line mass spectrometry peak area. These on-line metabolomics univariate models performed comparably to vibrational spectroscopy multivariate partial least squares regressions models reported in the literature, which typically are much more complex and time consuming to build. In conclusion, this work shows how on-line metabolomics can be used to directly monitor many bioprocess compounds of interest and obtain rich biological and bioprocess data.

Metabolome Profiling in the Plasma of Dogs with Idiopathic Dilated Cardiomyopathy: A Multiplatform Mass-Spectrometry-Based Approach.

Dilated cardiomyopathy is one of the important diseases in dogs and humans. The second most common cause of heart failure in dogs is idiopathic dilated cardiomyopathy (iDCM), which results in heart failure or sudden cardiac death due to arrhythmia. This study aimed to determine changes in the plasma metabolome of dogs with iDCM compared to healthy dogs. For that purpose, a multiplatform mass-spectrometry-based approach was used. In this study, we included two groups of dogs: 12 dogs with iDCM and 8 healthy dogs. A total of 272 metabolites were detected in the plasma samples of dogs by combining three approaches but four MS-based platforms (GC-MS, LC-MS (untargeted), LC-MS (targeted), and FIA-MS (targeted) methods). Our findings demonstrated changes in the canine plasma metabolome involved in the development of iDCM, including the different concentrations of amino acids, biogenic amines, acylcarnitines, triglycerides and diglycerides, sphingomyelins, and organic acids. The results of this study will enable the detection and monitoring of pathophysiological mechanisms involved in the development of iDCM in the future.

TopNEXt: automatic DDA exclusion framework for multi-sample mass spectrometry experiments.

MOTIVATION: Liquid Chromatography Tandem Mass Spectrometry experiments aim to produce high-quality fragmentation spectra, which can be used to annotate metabolites. However, current Data-Dependent Acquisition approaches may fail to collect spectra of sufficient quality and quantity for experimental outcomes, and extend poorly across multiple samples by failing to share information across samples or by requiring manual expert input. RESULTS: We present TopNEXt, a real-time scan prioritization framework that improves data acquisition in multi-sample Liquid Chromatography Tandem Mass Spectrometry metabolomics experiments. TopNEXt extends traditional Data-Dependent Acquisition exclusion methods across multiple samples by using a Region of Interest and intensity-based scoring system. Through both simulated and lab experiments, we show that methods incorporating these novel concepts acquire fragmentation spectra for an additional 10% of our set of target peaks and with an additional 20% of acquisition intensity. By increasing the quality and quantity of fragmentation spectra, TopNEXt can help improve metabolite identification with a potential impact across a variety of experimental contexts. AVAILABILITY AND IMPLEMENTATION: TopNEXt is implemented as part of the ViMMS framework and the latest version can be found at https://github.com/glasgowcompbio/vimms. A stable version used to produce our results can be found at 10.5281/zenodo.7468914.

Simulated-to-real benchmarking of acquisition methods in untargeted metabolomics.

Data-Dependent and Data-Independent Acquisition modes (DDA and DIA, respectively) are both widely used to acquire MS2 spectra in untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) metabolomics analyses. Despite their wide use, little work has been attempted to systematically compare their MS/MS spectral annotation performance in untargeted settings due to the lack of ground truth and the costs involved in running a large number of acquisitions. Here, we present a systematic in silico comparison of these two acquisition methods in untargeted metabolomics by extending our Virtual Metabolomics Mass Spectrometer (ViMMS) framework with a DIA module. Our results show that the performance of these methods varies with the average number of co-eluting ions as the most important factor. At low numbers, DIA outperforms DDA, but at higher numbers, DDA has an advantage as DIA can no longer deal with the large amount of overlapping ion chromatograms. Results from simulation were further validated on an actual mass spectrometer, demonstrating that using ViMMS we can draw conclusions from simulation that translate well into the real world. The versatility of the Virtual Metabolomics Mass Spectrometer (ViMMS) framework in simulating different parameters of both Data-Dependent and Data-Independent Acquisition (DDA and DIA) modes is a key advantage of this work. Researchers can easily explore and compare the performance of different acquisition methods within the ViMMS framework, without the need for expensive and time-consuming experiments with real experimental data. By identifying the strengths and limitations of each acquisition method, researchers can optimize their choice and obtain more accurate and robust results. Furthermore, the ability to simulate and validate results using the ViMMS framework can save significant time and resources, as it eliminates the need for numerous experiments. This work not only provides valuable insights into the performance of DDA and DIA, but it also opens the door for further advancements in LC-MS/MS data acquisition methods.

Proteomic changes associated with maternal dietary low ω6:ω3 ratio in piglets supplemented with seaweed Part II: Ileum proteomes.

This study evaluates how long-term dietary low ω6:ω3 ratio in sows and offspring's seaweed (SW) intake affects piglet intestinal function and growth through modifying ileum proteome. Sows were assigned to either control diet (CR, ω6:ω3 ratio = 13:1) or treatment diet (LR, ω6:ω3 = 4:1) during gestation and lactation (n = 8 each). The male weaned offspring were received a basal diet with or without SW powder supplementation (4 g/kg) for 21 days, denoted as SW and CT groups, respectively. In total, four groups of weaned piglets were formed following maternal and offspring's diets combination, represented by CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet ileum tissue was collected on day 22 post-weaning and analysed using TMT-based quantitative proteomics. The differentially abundant proteins (n = 300) showed the influence of maternal LR diet on protein synthesis, cell proliferation, and cell cycle regulation. In contrast, the SW diet lowered the inflammation severity and promoted ileal tissue development in CRSW piglets but reduced the fat absorption capacity in LRSW piglets. These results uncovered the mechanism behind the anti-inflammation and intestinal-boosting effects of maternal LR diet in piglets supplemented with SW.

Proteomic changes associated with maternal dietary low ω6:ω3 ratio in piglets supplemented with seaweed. Part I: Serum proteomes.

This study examines whether maternal low ω6:ω3 ratio diet and offspring SW supplementation can improve offspring immunity and performance by elucidating the effects on piglet serum proteome. A total of 16 sows were given either a standard (CR, 13:1) or low ω6:ω3 ratio diet (LR, 4:1) during pregnancy and lactation and their male weaned piglets were supplemented with SW powder (4 g/kg, SW) or not (CT) in a 21-day post-weaning (PW) diet. Four PW piglet groups were then identified based on dam and piglet treatment, namely CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet serum collected at weaning and d21 PW were analysed (n = 5 each) using TMT-based quantitative proteomics and validated by appropriate assays. The differentially abundant proteins (n = 122) displayed positive effects of maternal LR diet on anti-inflammatory properties and innate immune stimulation. Progeny SW diet activated the innate immunity and enhance the host defence during inflammation. These data demonstrate the value of decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet to boost their immunity and anti-inflammation properties. SIGNIFICANCE: This novel proteomic study in post-weaned piglets addresses the interplay between maternal and offspring nutritional interventions in a context of rapid and dynamic alterations in piglet metabolic status around weaning. Decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet can boost their immunity and anti-inflammation properties. This study also provides new insights into piglet serum proteome regulation during post-weaning, a critical development period in swine.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum.

The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.

Abundance of plasma proteins in response to divergent ratios of dietary ω6:ω3 fatty acids in gestating and lactating sows using a quantitative proteomics approach.

This study aimed to investigate the characteristic proteomic pattern of plasma from sows supplemented with low dietary ω6:ω3 fatty acids (FAs) ratio during gestation and lactation. Two dietary treatments (n = 8 each) comprised either a control ratio of ω6:ω3 FAs (CR, 13:1 during gestation and 10:1 during lactation) or a low ratio (LR, 4:1 during gestation and lactation) by adding soybean oil or linseed oil, respectively. High-resolution mass spectrometry-based quantitative proteomics was applied on plasma (n = 5 each) at day 108 of gestation (G108) and at the end of lactation (L-End), and a total of 379 proteins and 202 master proteins were identified. Out of these, four differentially abundant proteins between LR and CR samples at G108 may relate to serine-type endopeptidase inhibitor activity. Differentially abundant proteins in L-End versus G108 (12 up-regulated and 10 down-regulated) were positively correlated with the events that regulate plasma lipoproteins, stimulus- and defence-responses. These findings demonstrate the benefit of increased dietary ω3 FAs in modifying proteins involved in protective mechanisms against increased stresses in key life cycle phases in pigs. In addition, proteome changes from late gestation to late lactation disclosed the underlying mechanism of pigs in response to reproduction-related stimuli. SIGNIFICANCE: This study aimed to provide a proteomics insight into the beneficial effects of maternal diet supplementation with a low ω6:ω3 fatty acids ratio, based on previously reported performance and zootechnical data. The results suggest that a low dietary ω6:ω3 fatty acids ratio could enhance the cellular defence mechanisms against increased stresses and in particular to oxidative stress in sows during gestation and lactation, as reflected in proteomic changes of haptoglobin (HP), alpha-1-antitrypsin (SERPINA1) and serum amyloid P-component (APCS). Furthermore, significantly changed proteome profiles in sow plasma between late gestation and lactation phases have been revealed for the first time. This finding identified the adaptation mechanisms of sows to changing physiological events during reproduction.

Limited Impact of the Protein Corona on the Cellular Uptake of PEGylated Zein Micelles by Melanoma Cancer Cells.

The formation of a protein layer "corona" on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy.

Multi Platforms Strategies and Metabolomics Approaches for the Investigation of Comprehensive Metabolite Profile in Dogs with Babesia canis Infection.

Canine babesiosis is an important tick-borne disease worldwide, caused by parasites of the Babesia genus. Although the disease process primarily affects erythrocytes, it may also have multisystemic consequences. The goal of this study was to explore and characterize the serum metabolome, by identifying potential metabolites and metabolic pathways in dogs naturally infected with Babesia canis using liquid and gas chromatography coupled to mass spectrometry. The study included 12 dogs naturally infected with B. canis and 12 healthy dogs. By combining three different analytical platforms using untargeted and targeted approaches, 295 metabolites were detected. The untargeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomics approach identified 64 metabolites, the targeted UHPLC-MS/MS metabolomics approach identified 205 metabolites, and the GC-MS metabolomics approach identified 26 metabolites. Biological functions of differentially abundant metabolites indicate the involvement of various pathways in canine babesiosis including the following: glutathione metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; cysteine and methionine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. This study confirmed that host-pathogen interactions could be studied by metabolomics to assess chemical changes in the host, such that the differences in serum metabolome between dogs with B. canis infection and healthy dogs can be detected with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Our study provides novel insight into pathophysiological mechanisms of B. canis infection.

Rapid Development of Improved Data-Dependent Acquisition Strategies.

Tandem mass spectrometry (LC-MS/MS) is widely used to identify unknown ions in untargeted metabolomics. Data-dependent acquisition (DDA) chooses which ions to fragment based upon intensities observed in MS1 survey scans and typically only fragments a small subset of the ions present. Despite this inefficiency, relatively little work has addressed the development of new DDA methods, partly due to the high overhead associated with running the many extracts necessary to optimize approaches in busy MS facilities. In this work, we first provide theoretical results that show how much improvement is possible over current DDA strategies. We then describe an in silico framework for fast and cost-efficient development of new DDA strategies using a previously developed virtual metabolomics mass spectrometer (ViMMS). Additional functionality is added to ViMMS to allow methods to be used both in simulation and on real samples via an Instrument Application Programming Interface (IAPI). We demonstrate this framework through the development and optimization of two new DDA methods that introduce new advanced ion prioritization strategies. Upon application of these developed methods to two complex metabolite mixtures, our results show that they are able to fragment more unique ions than standard DDA strategies.

Proteomic analysis reveals differentially abundant proteins probably involved in the virulence of amastigote and promastigote forms of Leishmania infantum.

Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.

Postmortomics: The Potential of Untargeted Metabolomics to Highlight Markers for Time Since Death.

The success of forensic investigations involving fatalities very often depends on the establishment of the correct timeline of events. Currently used methods for estimating the postmortem interval (PMI) are mostly dependent on the professional and tacit experience of the investigator, and often with poor reliability in the absence of robust biological markers. The aim of this study was to investigate the potential of metabolomic approaches to highlight molecular markers for PMI. Rat and human muscle tissues, collected at various times postmortem, were analyzed using an untargeted metabolomics approach. Levels of certain metabolites (skatole, xanthine, n-acetylneuraminate, 1-methylnicotinamide, choline phosphate, and uracil) as well as most proteinogenic amino acids increased steadily postmortem. Threonine, tyrosine, and lysine show the most predictable evolution over the postmortem period, and may thus have potential for possible PMI markers in the future. This study demonstrates how a biomarker discovery approach can be extended to forensic investigations using untargeted metabolomics.

Functional colour genes and signals of selection in colour-polymorphic salamanders.

Coloration has been associated with multiple biologically relevant traits that drive adaptation and diversification in many taxa. However, despite the great diversity of colour patterns present in amphibians the underlying molecular basis is largely unknown. Here, we use insight from a highly colour-variable lineage of the European fire salamander (Salamandra salamandra bernardezi) to identify functional associations with striking variation in colour morph and pattern. The three focal colour morphs-ancestral black-yellow striped, fully yellow and fully brown-differed in pattern, visible coloration and cellular composition. From population genomic analyses of up to 4,702 loci, we found no correlations of neutral population genetic structure with colour morph. However, we identified 21 loci with genotype-phenotype associations, several of which relate to known colour genes. Furthermore, we inferred response to selection at up to 142 loci between the colour morphs, again including several that relate to coloration genes. By transcriptomic analysis across all different combinations, we found 196 differentially expressed genes between yellow, brown and black skin, 63 of which are candidate genes involved in animal coloration. The concordance across different statistical approaches and 'omic data sets provide several lines of evidence for loci linked to functional differences between colour morphs, including TYR, CAMK1 and PMEL. We found little association between colour morph and the metabolomic profile of its toxic compounds from the skin secretions. Our research suggests that current ecological and evolutionary hypotheses for the origins and maintenance of these striking colour morphs may need to be revisited.

In Silico Optimization of Mass Spectrometry Fragmentation Strategies in Metabolomics.

Liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) is widely used in identifying small molecules in untargeted metabolomics. Various strategies exist to acquire MS/MS fragmentation spectra; however, the development of new acquisition strategies is hampered by the lack of simulators that let researchers prototype, compare, and optimize strategies before validations on real machines. We introduce Virtual Metabolomics Mass Spectrometer (ViMMS), a metabolomics LC-MS/MS simulator framework that allows for scan-level control of the MS2 acquisition process in silico. ViMMS can generate new LC-MS/MS data based on empirical data or virtually re-run a previous LC-MS/MS analysis using pre-existing data to allow the testing of different fragmentation strategies. To demonstrate its utility, we show how ViMMS can be used to optimize N for Top-N data-dependent acquisition (DDA) acquisition, giving results comparable to modifying N on the mass spectrometer. We expect that ViMMS will save method development time by allowing for offline evaluation of novel fragmentation strategies and optimization of the fragmentation strategy for a particular experiment.

High-throughput quantification of carboxymethyl lysine in serum and plasma using high-resolution accurate mass Orbitrap mass spectrometry.

BACKGROUND: Carboxymethyl lysine is an advanced glycation end product of interest as a potential biomarker of cardiovascular and other diseases. Available methods involve ELISA, with potential interference, or isotope dilution mass spectrometry (IDMS), with low-throughput sample preparation. METHODS: A high-throughput sample preparation method based on 96-well plates was developed. Protein-bound carboxymethyl lysine and lysine were quantified by IDMS using reversed phase chromatography coupled to a high-resolution accurate mass Orbitrap Exactive mass spectrometer. The carboxymethyl lysine concentration (normalized to lysine concentration) was measured in 1714 plasma samples from the British Regional Heart Study (BRHS). RESULTS: For carboxymethyl lysine, the lower limit of quantification (LLOQ) was estimated at 0.16 µM and the assay was linear between 0.25 and 10 µM. For lysine, the LLOQ was estimated at 3.79 mM, and the assay was linear between 2.5 and 100 mM. The intra-assay coefficient of variation was 17.2% for carboxymethyl lysine, 9.3% for lysine and 10.5% for normalized carboxymethyl lysine. The inter-assay coefficient of variation was 18.1% for carboxymethyl lysine, 14.8 for lysine and 16.2% for normalized carboxymethyl lysine. The median and inter-quartile range of all study samples in each batch were monitored. A mean carboxymethyl lysine concentration of 2.7 µM (IQR 2.0-3.2 µM, range 0.2-17.4 µM) and a mean normalized carboxymethyl lysine concentration of 69 µM/M lysine (IQR 54-76 µM/M, range 19-453 µM/M) were measured in the BRHS. CONCLUSION: This high-throughput sample preparation method makes it possible to analyse large cohorts required to determine the potential of carboxymethyl lysine as a biomarker.

Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in Leishmania parasites.

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3' untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana.

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana.

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.