Bio21 - Theses
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ItemThe role of de novo myo-inositol synthesis and matabolism in Leishmania parasites( 2018)Leishmania are protozoan parasites responsible for a spectrum of sandfly-borne diseases known as the leishmaniases that cause substantial morbidity and mortality in some of the most impoverished countries in the world. Conventional anti leishmanials are limited by toxicity, high cost, and increasing resistance, highlighting an urgent need to identify novel drug targets. Leishmania parasites require the nutrient myo-inositol for a multitude of crucial cellular processes that relate to its role as an integral building block for inositol lipids, including phosphatidylinositol (PI), inositol phosphorylceramide (IPC), free and anchoring glycosylphosphatidylinositol (GPI), and organelle-specific phosphoinositides. This work investigates the extent to which Leishmania parasites are dependent on de novo myo-inositol synthesis versus salvage from the extracellular milieu both in vitro and in vivo for growth and pathogenesis. By generating defined genetic deletion mutants in Leishmania mexicana lacking two key putative enzymes in this pathway, namely myo inositol 3-phosphate synthase (INO1) and myo-inositol 3 phosphate monophosphatase 1 (IMP1), the importance of de novo myo-inositol synthesis was directly addressed. As expected, the L. mexicana ∆ino1 mutant lacked the capacity for de novo myo inositol synthesis and was an inositol auxotroph. Strikingly, this mutant could only grow in ex vivo infected macrophages which had been supplied with an exogenous sources of myo-inositol and was avirulent in the susceptible BALB/c mouse model, suggesting that myo-inositol levels in Leishmania-induced granulomas are low. Analysis of the steady state polar metabolite profile of ∆ino1 mutant and 13C glucose labelling experiments failed to reveal key differences compared to wild type, demonstrating that L. mexicana does not enter self preserving quiescence in response to myo-inositol starvation. This, combined with the profound changes in lipid composition revealed by lipidomic analysis, may underlie the rapid death phenotype induced by myo inositol starvation. A second L. mexicana mutant lacking IMP1 was also generated. Unexpectedly, deletion of imp1 did not lead to disruption of de novo myo-inositol synthesis, but resulted in a mutant that displayed a modest growth defect in vitro that was unrelated to exogenous myo inositol levels. Despite the presence of de novo synthesis, the Δimp1 mutant was unable to proliferate in ex vivo macrophages or cause disease in mice. Global metabolomic and lipidomic analysis of this mutant revealed subtle changes in central carbon metabolism and lipid profile. Further studies showed that the loss of virulence of this mutant was linked to increased sensitivity to elevated temperatures encountered in the mammalian host, suggesting an unexpected role for IMP1 in the development of thermotolerance and differentiation to the amastigote stage. Genomic analysis also revealed a second putative IMPase isoform, IMP2, which may explain why loss of IMP1 does not lead to pertrubation of de novo myo-inositol synthesis in the Δimp1 mutant. In summary, this work highlights the importance of de novo myo-inositol synthesis for Leishmania growth and survival, providing a better understanding of a vital pathway. This improved understanding of the metabolic mechanisms essential for Leishmania provides further insights into their pathogenesis and validates enzymes in this pathway as new targets for the development of new strategies to combat the rising drug resistance.
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ItemMetabolomics reveals the relationship between the host and the gut in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome( 2017)Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disorder that likely has a complex etiological mechanism entailing predisposing, triggering, and maintaining factors. ME/CFS patients present with incapacitating long-term post-exertional malaise and a broad array of neurological, immunological and gastrointestinal symptoms. Few studies have looked to detect the basis of the energy depletion and the gastrointestinal symptoms; both are observable by metabolomics. Metabolomics is the study of metabolites, any small organic molecule, including amino acids, sugars, lipids and more. Measuring metabolites in biofluids has been performed for decades, but the development of fast, automated and standardised methods has led to the “-omics” suffix. Previous metabolite studies on ME/CFS have revealed alterations that presented some clues of amino acid and oxidative stress disturbance in ME/CFS. These studies were not using standardised metabolomics techniques and added no gastrointestinal context. In this thesis, the first application of metabolomics to study ME/CFS is described from an initial preliminary study on blood to a larger study on host metabolite changes and their association with gut metabolites and bacteria. All metabolomics research in this thesis was conducted using NMR spectroscopy. The preliminary study was on blood samples from 11 ME/CFS and 10 non-ME/CFS subjects. Glutamine and ornithine were found to be reduced in ME/CFS, and correlations of these metabolites with other metabolites revealed relationships existing between glucogenic amino acids and metabolites that participate in the urea cycle. The second study was on a much larger all-female cohort, 34 ME/CFS and 25 non-ME/CFS participants. Participants donated faeces, urine and blood samples in a timeframe for associations between biofluids to be made. In ME/CFS patients, the metabolite changes indicated a decreased glycolysis (or increased gluconeogenesis) was occurring. We surmised that this was reducing the adequate supply of acetyl-CoA to the citric acid cycle and proposed that amino acids were being increasingly utilised instead, as evidenced by a reduction in blood and urine amino acids. The prominent decrease in blood glutamate and increase in blood aspartate highlighted that aspartate transaminase (AST) might be upregulated to allow amino acids to fuel the TCA cycle. Further evidence for reduced glycolysis was an increase in creatinine, an alternate resource for anaerobic ATP production within muscle. A reperfusion pathway that produces superoxide and results in urinary allantoin accumulation was also observed in ME/CFS patients adding more evidence for increased oxidative stress. In the faeces, Clostridium spp. was increased and Bacteroides spp. were decreased, possibly indicating an alteration in microbes in ME/CFS was towards a scavenging phenotype. The faecal metabolites changes highlighted an increase in fermentation was occurring in the colon of ME/CFS producing SCFA accumulation. Furthermore, the increase in isovalerate suggests that amino acids were increasingly being used for fermentation in ME/CFS patients. We propose that the increasing use of amino acids for ATP production in the host has reduced the production of digestive enzymes, gut bacteria are provided with increased dietary substrate and increased provisions of amino acids in the colon result in increased SCFA and isovalerate via fermentation.