Eleanor C. Saunders

Isotopomer Profiling of Leishmania mexicana Promastigotes Reveals Important Roles for Succinate Fermentation and Aspartate Uptake in Tricarboxylic Acid Cycle (TCA) Anaplerosis, Glutamate Synthesis, and Growth

Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains poorly defined. In this study, we have followed the incorporation of various 13C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and 13C NMR. [U-13C]Glucose was rapidly incorporated into intermediates in glycolysis, the pentose phosphate pathway, and the cytoplasmic carbohydrate reserve material, mannogen. Enzymes involved in the upper glycolytic pathway are sequestered within glycosomes, and the ATP and NAD+ consumed by these reactions were primarily regenerated by the fermentation of phosphoenolpyruvate to succinate (glycosomal succinate fermentation). The initiating enzyme in this pathway, phosphoenolpyruvate carboxykinase, was exclusively localized to the glycosome. Although some of the glycosomal succinate was secreted, most of the C4 dicarboxylic acids generated during succinate fermentation were further catabolized in the TCA cycle. A high rate of TCA cycle anaplerosis was further suggested by measurement of [U-13C]aspartate and [U-13C]alanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.

Induction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

Central carbon metabolism of Leishmania parasites.

Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.

A dynamic programming approach for the alignment of signal peaks in multiple gas chromatography-mass spectrometry experiments.

BackgroundGas chromatography-mass spectrometry (GC-MS) is a robust platform for the profiling of certain classes of small molecules in biological samples. When multiple samples are profiled, including replicates of the same sample and/or different sample states, one needs to account for retention time drifts between experiments. This can be achieved either by the alignment of chromatographic profiles prior to peak detection, or by matching signal peaks after they have been extracted from chromatogram data matrices. Automated retention time correction is particularly important in non-targeted profiling studies.ResultsA new approach for matching signal peaks based on dynamic programming is presented. The proposed approach relies on both peak retention times and mass spectra. The alignment of more than two peak lists involves three steps: (1) all possible pairs of peak lists are aligned, and similarity of each pair of peak lists is estimated; (2) the guide tree is built based on the similarity between the peak lists; (3) peak lists are progressively aligned starting with the two most similar peak lists, following the guide tree until all peak lists are exhausted. When two or more experiments are performed on different sample states and each consisting of multiple replicates, peak lists within each set of replicate experiments are aligned first (within-state alignment), and subsequently the resulting alignments are aligned themselves (between-state alignment). When more than two sets of replicate experiments are present, the between-state alignment also employs the guide tree. We demonstrate the usefulness of this approach on GC-MS metabolic profiling experiments acquired on wild-type and mutant Leishmania mexicana parasites.ConclusionWe propose a progressive method to match signal peaks across multiple GC-MS experiments based on dynamic programming. A sensitive peak similarity function is proposed to balance peak retention time and peak mass spectra similarities. This approach can produce the optimal alignment between an arbitrary number of peak lists, and models explicitly within-state and between-state peak alignment. The accuracy of the proposed method was close to the accuracy of manually-curated peak matching, which required tens of man-hours for the analyzed data sets. The proposed approach may offer significant advantages for processing of high-throughput metabolomics data, especially when large numbers of experimental replicates and multiple sample states are analyzed.

LeishCyc: a biochemical pathways database for Leishmania major

BackgroundLeishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen.DescriptionThe LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2), based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute).ConclusionThe LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania biochemical networks and is a tool for analysis, interpretation, and visualization of Leishmania Omics data (transcriptomics, proteomics, metabolomics) in the context of metabolic pathways. LeishCyc is the first such database for the Trypanosomatidae family, which includes a number of other important human parasites. Flexible query/visualization capabilities are provided by the Pathway Tools software and its Web interface. The LeishCyc database is made freely available over the Internet http://www.leishcyc.org.

Characterization of Metabolically Quiescent Leishmania Parasites in Murine Lesions Using Heavy Water Labeling

Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters in vivo. Here, we have measured the replication and physiological state of Leishmania mexicana parasites in murine inflammatory lesions using 2H2O labeling. Infected BALB/c mice were labeled with 2H2O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from ex vivo infected macrophages or cultured in vitro, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the Leishmania response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions in situ.

Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?

A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments. How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.

Leishmania major Methionine Sulfoxide Reductase A Is Required for Resistance to Oxidative Stress and Efficient Replication in Macrophages

Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H2O2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H2O2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.

Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages

Abstract Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.

High-content assay for measuring intracellular growth of Leishmania in human macrophages.

Leishmania species are sandfly-transmitted protozoan parasites that cause a spectrum of diseases, ranging from localized skin lesions to fatal visceral disease, in more than 12 million people worldwide. These parasites primarily target macrophages in their mammalian hosts and proliferate as non-motile amastigotes in the phagolysosomal compartment of these cells. High-throughput screens for measuring Leishmania growth within this intracellular niche are needed to identify host and parasite factors that are required for virulence and to identify new drug candidates. Here we describe the development of a new high-content imaging method for quantifying the intracellular growth of Leishmania mexicana parasites in THP-1 macrophages. Wild-type parasites were pre-stained with the fluorescent dye CellTracker(™) Orange CMRA and used to infect THP-1 macrophages in 384-well plates. Infected and uninfected macrophages were subsequently stained with CellTracker Green CMFDA, allowing accurate quantitation of the number of parasites per macrophage using separate detector channels. We validated this method for use in high-content drug screening by examining the dose dependence of known anti-leishmanial drugs on intracellular growth. Unlike previous protocols, this method does not require the generation of transgenic fluorescent or bioluminescent parasite lines and can be readily adapted for screening different Leishmania species, strains, or mutant lines in a wide range of phagocytic host cell types.