Metabolism and drug resistance in Trypanosomatids

Wildridge, David (2012) Metabolism and drug resistance in Trypanosomatids. PhD thesis, University of Glasgow.

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Abstract

The principle aim of this project is the investigation of metabolism and mechanisms of pentamidine resistance in trypanosomatids. An understanding of these mechanisms may allow the development of novel drugs to treat Leishmaniasis and human African trypanosomiasis (HAT), caused by the protozoan parasites Leishmania spp and Trypanosoma brucei. In this study a
pentamidine resistance L. mexicana promastigote cell line was generated in vitro. This cell line was 20-fold resistant to pentamidine when compared to the parental wild type cells. Furthermore, these lines were cross resistant to other diamidine compounds. A proteomic analysis of these cell lines revealed numerous changes to the proteome, with the down regulation of several flagellar proteins. A hypothesis to investigate a role of the voltage dependent anion channel (VDAC) in pentamidine resistance was also explored. The metabolomic approach involved the investigation of transketolase and the pentose phosphate pawthway. A previous study involving a transketolase knockout T. brucei cell line indicated that an increased sensitivity to pentamidine and methylene blue. A transketolase deficient L. mexicana cell line was generated to test this hypothesis in Leishmania, however the differences were minimal. A metabolomic analysis of the L. mexicana tkt null cell line (lmtkt-/-) revealed an increase in ribose 5-phosphate, a key substrate of transketolase. Erythrose 4-phosphate also increased in the lmtkt-/- cells, indicating a source of this metabolite independent of TKT. It appears that the deletion of TKT prevents any flux through the oxidative branch of the PPP returning to the glycolytic pathway. Interestingly, the lmtkt-/- cells do not acidify the medium to the same extent as the wild type cells; however a glucose assay indicated that both cell lines used similar quantities of glucose. This would suggest that there is a change in the metabolites excreted by the lmtkt-/- cell line. Finally, a global metabolomics
approach was investigated using high resolution mass spectrometry. Metabolomics is a rapidly developing field in systems biology, and whilst significant improvements have been made in mass spectrometry; the ability to analyse and interpret raw metabolomic datasets on a global scale has been largely neglected. Consequently, a database program to query these complex
datasets was constructed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: Barrett, Prof. Michael P. and Burchmore, Dr. Richard
Date of Award: 2012
Depositing User: Mr David Wildridge
Unique ID: glathesis:2012-3622
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 15 Jan 2013 13:35
Last Modified: 10 Dec 2015 08:32
URI: http://theses.gla.ac.uk/id/eprint/3622

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