Phenotypic characterisation of glucose transporter knockout Leishmania mexicana

Lamasudin, Dhilia Ude (2012) Phenotypic characterisation of glucose transporter knockout Leishmania mexicana. PhD thesis, University of Glasgow.

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Abstract

The aim of this project was to investigate nutrient acquisition by glucose transporter null mutant Leishmania mexicana, to understand how genetic ablation of glucose transporter genes in these protozoan parasites affects their phenotype. It has previously been demonstrated that glucose transporter null mutant Leishmania (ΔLmGT) promastigotes have lost the capacity to transport glucose as well other hexoses. Glucose is a potential carbon source for promastigotes, which are typically maintained in glucose-rich media and which encounter high levels of glucose in the sandfly midgut. Despite inability to transport glucose, the null mutant parasite is able to survive and grow, although at a reduced rate in media that contains non-carbohydrate potential carbon sources. This finding suggesting that the null mutant may utilise other carbon sources. In this project, a variety of approaches have been applied to investigate the nature of these alternative carbon source and the mechanisms by which they are acquired and metabolised. Amino acid uptake assays revealed that glucose transporter null mutant promastigotes take up several key amino acids at a significantly enhanced rate, compared with wild type promastigotes. A comparative metabolomic analysis was applied for more comprehensive comparison between the wild type and glucose transporter null mutant. Though the uptake assay and metabolomic analysis showed distinct changes in nutrient uptake and metabolisms between the two cell lines, these results does not tell whether the changes are due to changes in transporter activity or expression, nor does it inform on the enzymes involved in these processess. Thus, a proteomic approach was applied. Since membrane transporter were a focus of interest, but are known to be highly hydrophobic and of relatively low abundance, it was necessary to develop protocols for membrane protein enrichment and fractionation in Leishmania mexicana. A previous comparative proteomic analysis, which did not represent membrane proteins, had highlighted changes in the abundance of a major component of the paraflagellar rod. This unexpected result was further investigated, by Western blotting, electron microscopy and taxis assay. The results point to changes in flagellar structure of function in glucose transporter null mutant Leishmania. It is no doubt the experiments carried out in this project have shown interesting changes in glucose transporter null mutant Leishmania, in terms of nutrient acquisition and phenotypic characteristics. The data provide a new set of ideas as to how Leishmania thrive in a condition where glucose is scarce. These data may have relevance to leishmaniasis in mammals, as it is likely that glucose is constitutively scarce in the intracellular environment inhabited by the amastigote. However, due to time constraints, it was not possible to extend these investigations to the amastigote stage of Leishmania.

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
Funder's Name: UNSPECIFIED
Supervisor's Name: Burchmore, Dr. Richard
Date of Award: 2012
Depositing User: Mrs Marie Cairney
Unique ID: glathesis:2012-4161
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 04 Apr 2013 11:51
Last Modified: 13 Oct 2017 16:14
URI: http://theses.gla.ac.uk/id/eprint/4161

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