Metabolomic approaches for the identification of metabolic pathways in Trypanosoma brucei

Johnston, Katharina Louise (2015) Metabolomic approaches for the identification of metabolic pathways in Trypanosoma brucei. PhD thesis, University of Glasgow.

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Abstract

Trypanosoma brucei is a parasitic protozoan that can cause human African trypanosomiasis (HAT) and Nagana in cattle. Human African trypanosomiasis is deadly when left untreated, and thus there is an urgent need to develop new drugs against this disease. As trypanosomes are early diverged eukaryotes, it is anticipated that studying their metabolism can identify novel drug targets. The main drug currently in use against the late encephalitic stage, Eflornithine, was shown to inhibit an essential pathway in trypanosomes (Yarlett and Bacchi, 1989). In this Thesis three approaches were used to apply metabolomic and proteomic techniques for protein function identification and to investigate metabolic pathways. The genome of T. brucei has been published (Berriman et al., 2005) and data is available via databases, such as TriTrypDB, a database dedicated to the trypanosomatids (Aslett et al., 2009). An estimated 40% of the identified genes in this organism are annotated with an unknown or putative function. In 2006, Saito et al. developed a systematic method to ascertain enzyme function based on an in vitro assay, in combination with metabolite profiling. This approach was successfully applied in several other studies. Here, I investigate the use of this method for its application in a high throughput approach for unknown enzyme identification in trypanosomes. Seven putative identified enzymes were randomly selected from TriTrypDB, cloned and expressed in E. coli and a function could be attributed to at least one of the enzymes. Furthermore, the amino acid metabolism in trypanosomes was investigated; using stable isotope labelling combined with metabolomics. The flux of labelled compounds could be traced through the organism showing the active metabolic pathways of L-methionine, L-proline and L-arginine in T. brucei. Two T. b. brucei strains used in this study, GVR35 and 427, cause different forms of infections in their mammalian host. GVR35 causes a chronic infection and invades the central nervous system (CNS) with varying parasitemia in mice, whereas infection with strain 427 presents an acute form with high parasitaemia, causing high mortality, without invading the CNS. What causes this difference in the progression of infection? Secreted or excreted proteins from the parasites, referred to as the secretome, have been described as being important factor for virulence and avoiding the host immune response (Geiger et al., 2010) and Garzon et al. (2006) showed that excreted/secreted proteins can inhibit the maturation of dentritic cells and stop them from inducing a lymphocytic allogenic response. Significant differences in proteins secreted from these two strains are discussed; although the results are preliminary.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Parasitology
Funder's Name: UNSPECIFIED
Supervisor's Name: Barrett, Professor Michael and Burchmore, Dr. Richard
Date of Award: 2015
Depositing User: Mrs Katharina Louise Johnston
Unique ID: glathesis:2015-6701
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 29 Sep 2015 10:43
Last Modified: 05 Oct 2015 09:28
URI: http://theses.gla.ac.uk/id/eprint/6701

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