Identification of metabolically quiescent Leishmania mexicana parasites in peripheral and cured dermal granulomas using stable isotope tracing imaging mass spectrometry

Leishmania 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 Leishmania mexicana in dermal granulomas in susceptible BALB/c mice, using in vivo heavy water labeling and ultra high-resolution imaging mass spectrometry. Quantitation of the rate of turnover of parasite and host-specific lipids at high spatial resolution, suggested that the granuloma core comprised mixed populations of metabolically active and quiescent parasites. Unexpectedly, a significant population of metabolically quiescent parasites was also identified in the surrounding collagen-rich, dermal mesothelium. Mesothelium-like tissues harboring quiescent parasites progressively replaced macrophage-rich granuloma tissues following treatment with the first-line drug, miltefosine. In contrast to the granulomatous tissue, neither the mesothelium nor newly deposited tissue sequestered miltefosine. These studies suggest that the presence of quiescent parasites in acute granulomatous tissues, together with the lack of miltefosine accumulation in cured lesion tissue, may contribute to drug failure and nonsterile cure. IMPORTANCE Many microbial pathogens switch between different growth and physiological states in vivo in order to adapt to local nutrient levels and host microbicidal responses. Heterogeneity in microbial growth and metabolism may also contribute to nongenetic mechanisms of drug resistance and drug failure. In this study, we have developed a new approach for measuring spatial heterogeneity in microbial metabolism in vivo using a combination of heavy water (2H2O) labeling and imaging mass spectrometry. Using this approach, we show that lesions contain a patchwork of metabolically distinct parasite populations, while the underlying dermal tissues contain a large population of metabolically quiescent parasites. Quiescent parasites also dominate drug-depleted tissues in healed animals, providing an explanation for failure of some first line drugs to completely eradicate parasites. This approach is broadly applicable to study the metabolic and growth dynamics in other host-pathogen interactions

Metabolomic analysis of trypanosomatid protozoa

Metabolomics aims to measure all low molecular weight chemicals within a given system in a manner analogous to transcriptomics, proteomics and genomics. In this review we highlight metabolomics approaches that are currently being applied to the kinetoplastid parasites, Trypanosoma brucei and Leishmania spp. The use of untargeted metabolomics approaches, made possible through advances in mass spectrometry and informatics, and stable isotope labelling has increased our understanding of the metabolism in these organisms beyond the views established using classical biochemical approaches. Set within the context of metabolic networks, predicted using genome-wide reconstructions of metabolism, new hypothesises on how to target aspects of metabolism to design new drugs against these protozoa are emergin

Acylation-dependent and-independent membrane targeting and distinct functions of small myristoylated proteins (SMPs) in Leishmania major

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function

Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii

Toxoplasma gondii proliferates within host cell vacuoles where the parasite relies on host carbon and nutrients for replication. To assess how T. gondii utilizes these resources, we mapped the carbon metabolism pathways in intracellular and egressed parasite stages. We determined that intracellular T. gondii stages actively catabolize host glucose via a canonical, oxidative tricarboxylic acid (TCA) cycle, a mitochondrial pathway in which organic molecules are broken down to generate energy. These stages also catabolize glutamine via the TCA cycle and an unanticipated γ-aminobutyric acid (GABA) shunt, which generates GABA and additional molecules that enter the TCA cycle. Chemically inhibiting the TCA cycle completely prevents intracellular parasite replication. Parasites lacking the GABA shunt exhibit attenuated growth and are unable to sustain motility under nutrient-limited conditions, suggesting that GABA functions as a short-term energy reserve. Thus, T. gondii tachyzoites have metabolic flexibility that likely allows the parasite to infect diverse cell types

Structures of glycosylphosphatidylinositol membrane anchors from Saccharomyces cerevisiae.

Metabolic labeling studies suggest that Saccharomyces cerevisiae contains many glycoproteins that are anchored in the lipid bilayer by glycosylphosphatidylinositol membrane anchors. Membrane anchors were purified from a crude yeast membrane protein fraction and analyzed by two-dimensional 1H-1H NMR, fast atom bombardment-mass spectrometry, compositional and methylation linkage analyses, as well as chemical and enzymatic modifications. The yeast glycosylphosphatidylinositol anchors consist of the following structures: ethanolamine-PO4-6(R-2)Man alpha 1-2Man alpha 1-6Man alpha 1-4Glc-NH2 alpha 1-6myo-inositol-1-PO4-lipid, where R is mainly Man alpha 1- (80%) with some Man alpha 1-2Man alpha 1- (15%) and Man alpha 1-3Man alpha 1- (5%). The core region of the yeast anchors (ethanolamine-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 alpha 1-6myo-inositol-1-PO4) is identical to the conserved core region found in glycosylphosphatidylinositol anchors from protozoa and mammals. The lipid moieties of the total yeast glycosylphosphatidylinositol anchors are mainly ceramides, consisting mostly of C18:0 phytosphingosine and C26:0 fatty acid. However, the lipid moiety of the glycosylphosphatidylinositol anchor of the purified ggp125 protein is a lyso- or diacylglycerol, containing C26:0 fatty acids. This suggests that yeast adds different lipid components to the glycosylphosphatidylinositol anchors of different proteins

Metabolomics provide sensitive insights into the impacts of low level environmental contamination on fish health—a pilot study

This exploratory study aims to investigate the health of sand flathead (Platycephalus bassensis) sampled from five sites in Port Phillip Bay, Australia using gas chromatography-mass spectrometry (GC-MS) metabolomics approaches. Three of the sites were the recipients of industrial, agricultural, and urban run-off and were considered urban sites, while the remaining two sites were remote from contaminant inputs, and hence classed as rural sites. Morphological parameters as well as polar and free fatty acid metabolites were used to investigate inter-site differences in fish health. Significant differences in liver somatic index (LSI) and metabolite abundance were observed between the urban and rural sites. Differences included higher LSI, an increased abundance of amino acids and energy metabolites, and reduced abundance of free fatty acids at the urban sites compared to the rural sites. These differences might be related to the additional energy requirements needed to cope with low-level contaminant exposure through energy demanding processes such as detoxification and antioxidant responses as well as differences in diet between the sites. In this study, we demonstrate that metabolomics approaches can offer a greater level of sensitivity compared to traditional parameters such as physiological parameters or biochemical markers of fish health, most of which showed no or little inter-site differences in the present study. Moreover, the metabolite responses are more informative than traditional biomarkers in terms of biological significance as disturbances in specific metabolic pathways can be identified

Analysis of inter-laboratory metabolomics experiments

Metabolomics Australia is developing standardised retention time locked GC-MS analyses methods that can be used in a laboratory independent manner and derive the same result. Achieving this level of reproducibility would allow for analysis of samples from large projects at multiple sites potentially increasing throughput substantially. To validate our methods we have prepared a standard mix containing ~40 known metabolites (mm). We have also prepared an additional version of this mix, where a selection of metabolites is spiked at a significantly higher abundance (mm+). Replicates (6-8) of the unspiked (mm) and spiked (mm+) metabolite mix were run on four different GC-MS machines at three laboratories using two GC-MS methods differing in temperature gradient. Target ion areas for the metabolites in the mix were extracted using Analyser Pro software (SpectralWorks) and presented as a matrix for statistical analysis. A linear model with an interaction term(s) is presented here to analyse and interpret these inter-laboratory metabolomics measurements. This model will give us a basis to build more complex models where different groups/types of samples from a single study are processed at different laboratories. This work is funded by Metabolomics Australi