Lipidomic investigations into the phospholipid content and metabolism of various kinetoplastids
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This work expands the knowledge on phospholipid metabolism in the kinetoplastid parasites: T. brucei, T. cruzi, Leishmania spp. that cause neglected tropical diseases and the related non-human pathogenic Crithidia fasiculata. As a close relative of parasitic kinetoplasts, specifically Leishmania, it is hypothesised that Crithidia fasiculata possesses a similar lipid biosynthetic capability and therefore represent an attractive model organism. Database mining the Crithidia genome revealed the ability to biosynthesise all of the main phospholipid species. Utilising various lipidomic techniques, a high level of an ω-6 18:3 fatty acid was observed, alongside an uncommon Δ19:0 fatty acid that was later identified to be exclusive attributed to PE species. Sphingolipid metabolism was shown to resemble that of Leishmania and T. cruzi, given the exclusive production of inositol-phosphoceramide species and no sphingomyelin species being observed. Using labelled precursors, Crithidia were seen to uptake and incorporate extracellular inositol into both phosphatidylinositol and inositol-phosphoceramide species. Crithidia were also shown to utilise both the Kennedy pathway and methylation of phosphatidylethanolamine to form phosphatidylcholine. The phospholipidome of T. cruzi revealed several phosphatidylserine species for the first time, suggesting a functional phosphatidylserine synthase. Current knowledge of T.cruzi sphingolipid biosynthesis was also confirmed as only inositol xxxi phosphoceramide species were observed. The identification and subsequent characterisation of novel phosphonolipid species are reported for the first time. Utilising lipidomic methodologies and labelled precursors, the relative contribution of the intracellular inositol pools within bloodstream and procyclic T. brucei towards PI biosynthesis was examined. This highlighted that the synthesis/turnover rates for specific phosphatidylinositol and inositol-phosphoceramide species are unequal. Efforts to optimise media conditions highlighted that under reduced levels of serum/glucose/inositol, bloodstream T. brucei unexpectedly adjusts its inositol metabolism. The procyclic parasite exemplifies this fact, as under inositol/glucose deficient media conditions they appear to have adapted to utilising glucogenesis and inositol de-novo synthesis. This work highlights that these parasites are rapidly dividing, their unique features of lipid metabolism may be exploitable for drug discovery purposes.
Thesis, PhD Doctor of Philosophy
Embargo Date: 2020-04-05
Embargo Reason: Thesis restricted in accordance with University regulations. Electronic copy restricted until 5th April 2020
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