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.

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 > School of Infection & Immunity
• 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: https://theses.gla.ac.uk/id/eprint/4161