Remodeling of Carbon Metabolism during Sulfoglycolysis in Escherichia coli.

Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E.

Oxidative desulfurization pathway for complete catabolism of sulfoquinovose by bacteria.

Catabolism of sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose), the ubiquitous sulfosugar produced by photosynthetic organisms, is an important component of the biogeochemical carbon and sulfur cycles. Here, we describe a pathway for SQ degradation that involves oxidative desulfurization to release sulfite and enable utilization of the entire carbon skeleton of the sugar to support the growth of the plant pathogen SQ or its glycoside sulfoquinovosyl glycerol are imported into the cell by an ATP-binding cassette transporter system with an associated SQ binding protein. A sulfoquinovosidase hydrolyzes the SQ glycoside and the liberated SQ is acted on by a flavin mononucleotide-dependent sulfoquinovose monooxygenase, in concert with an NADH-dependent flavin reductase, to release sulfite and 6-oxo-glucose.

Metabolic stringent response in intracellular stages of Leishmania.

Leishmania are unusual in being able to survive long-term in the mature phagolysosome compartment of macrophages and other phagocytic cells in their mammalian hosts. Key to their survival in this niche, Leishmania amastigotes switch to a slow growth state and activate a stringent metabolic response. The stringent metabolic response may be triggered by multiple stresses and is associated with decreased metabolic fluxes, restricted use of sugars and fatty acids as carbon sources and increased dependence on metabolic homeostasis pathways.

Identification of Metabolically Quiescent Parasites in Peripheral and Cured Dermal Granulomas Using Stable Isotope Tracing Imaging Mass Spectrometry.

are sandfly-transmitted protists that induce granulomatous lesions in their mammalian host. Although infected host cells in these tissues can exist in different activation states, the extent to which intracellular parasites stages also exist in different growth or physiological states remains poorly defined. Here, we have mapped the spatial distribution of metabolically quiescent and active subpopulations of in dermal granulomas in susceptible BALB/c mice, using heavy water labeling and ultra high-resolution imaging mass spectrometry.

Immunometabolism of Leishmania granulomas.

Leishmania are parasitic protists that cause a spectrum of diseases in humans characterized by the formation of granulomatous lesions in the skin or other tissues, such as liver and spleen. The extent to which Leishmania granulomas constrain or promote parasite growth is critically dependent on the host T-helper type 1/T-helper type 2 immune response and the localized functional polarization of infected and noninfected macrophages toward a classically (M1) or alternatively (M2) activated phenotype. Recent studies have shown that metabolic reprograming of M1 and M2 macrophages underpins the capacity of these cells to act as permissive or nonpermissive host reservoirs, respectively.

Publisher Correction: Metabolic characteristics of CD8 T cell subsets in young and aged individuals are not predictive of functionality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic characteristics of CD8 T cell subsets in young and aged individuals are not predictive of functionality.

Virtual memory T (T) cells are antigen-naïve CD8 T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (T) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of T cells and their altered functionality with age, here we investigate T cell metabolism and its association with longevity and functionality.

Leishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models.

Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L.

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.

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

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).

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.

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.

Use of (13)C stable isotope labelling for pathway and metabolic flux analysis in Leishmania parasites.

This protocol describes the combined use of metabolite profiling and stable isotope labelling to define pathways of central carbon metabolism in the protozoa parasite, Leishmania mexicana. Parasite stages are cultivated in standard or completely defined media and then rapidly transferred to chemically equivalent media containing a single (13)C-labelled nutrient. The incorporation of label can be followed over time or after establishment of isotopic equilibrium by harvesting parasites with rapid metabolic quenching.

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 (13)C-stable isotope resolved metabolomics and (2)H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state.

Membrane targeting of the small myristoylated protein 2 (SMP-2) in Leishmania major.

Leishmania parasites express three highly conserved small myristoylated proteins (SMPs) that are targeted to distinct membranes. SMP-1 is exclusively found in the flagellum, depending on myristoylation and palmitoylation. In contrast, monoacylated SMP-2 and SMP-4 are localized to the flagellar pocket and plasma membrane, respectively.

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.

LeishCyc: a guide to building a metabolic pathway database and visualization of metabolomic data.

The complexity of the metabolic networks in even the simplest organisms has raised new challenges in organizing metabolic information. To address this, specialized computer frameworks have been developed to capture, manage, and visualize metabolic knowledge. The leading databases of metabolic information are those organized under the umbrella of the BioCyc project, which consists of the reference database MetaCyc, and a number of pathway/genome databases (PGDBs) each focussed on a specific organism.

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 (13)C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and (13)C NMR.

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

Background: Gas 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.

Progressive peak clustering in GC-MS Metabolomic experiments applied to Leishmania parasites.

Motivation: A common problem in the emerging field of metabolomics is the consolidation of signal lists derived from metabolic profiling of different cell/tissue/fluid states where a number of replicate experiments was collected on each state.

Results: We describe an approach for the consolidation of peak lists based on hierarchical clustering, first within each set of replicate experiments and then between the sets of replicate experiments. The problems of finding the dendrogram tree cutoff which gives the optimal number of peak clusters and the effect of different clustering methods were addressed.