There is an urgent need for new chemotherapies against human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei. It is anticipated that the parasites’ divergent biochemistry will enable development of novel therapies. To study the behaviour of a complex network as metabolism, one can employ mathematical models. In this thesis, metabolism of bloodstream form T. brucei was investigated. Cellular metabolism consists as a complex system connecting enzymes with metabolites, and to study such a network one can construct mathematical models that describe the connections within the biological system. A previously published, and well-curated model of glycolysis in bloodstream form T. brucei (Bakker BM, et al. (1997) J Biol Chem 272:3207 15), was extended here with the pentose phosphate pathway (PPP), the second major pathway in glucose metabolism in most life forms. Several hypotheses were derived during the model building process and these were tested experimentally. It became apparent that the glycosomal bound-phosphate balance is essential for the parasite. Extension of the glycolytic model with the PPP introduced the risk of a so-called ’phosphate leak’, where bound-phosphates are depleted in the glycosome. Two hypotheses were investigated in silico, while one hypothesis could also be tested experimentally; (i) a glycosomal ATP:ADP antiporter was proposed, but in silico analysis indicated that the activity of such an antiporter requires tight regulation. (ii) A glycosomal ribokinase was investigated both in silico and experimentally. Genetic mutants indicated that ribokinase is essential to bloodstream form T. brucei, albeit at low levels. Additional analysis of the generated models indicated that ablation of 6-phosphogluconate dehydrogenase (6PGDH) in T. brucei is lethal by a different mechanism as seen in other organisms. Overall, extension of the glycolytic model with the PPP demonstrated the fragility of the model regarding the bound-phosphate balance and indicated that future analysis on glycosomal metabolism should be focused on this. Important in the use of mathematical models of metabolism is that the underlying stoichiometry of the model reflects (albeit with simplification) the in vivo system. It is therefore paramount to know what enzyme activities are present in the organism of interest. In this thesis a metabolomics approach was used to elucidate the function of three T. brucei genes. These genes were putatively annotated as arginase (ARG), N-acetylornithine deacetylase (NAO) and nicotinamidase (NAM). The results suggested that ARG has catalytic activity as tryptophan monooxygenase (EC 1.3.12.3), while substrate promiscuity was indicated for NAO and NAM. The work presented in this thesis has provided us with new insights on trypanosomal metabolism. The extended model now allows us to research a larger part of T. brucei metabolism with mathematical modelling, and will thereby aid in the identification and further investigation of (proposed) drug targets.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:566436 |
| Date | January 2012 |
| Creators | Kerkhoven, Eduard Johannes |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/3879/ |
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