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Miller, Thomas (2020) Exploring the surface of Trypanosoma brucei through protein sorting and recombinant expression. PhD thesis, University of Nottingham.

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Abstract

Surface membrane structure and composition define the biological niche of a unicellular organism, controlling how it interacts with and survives in its environment. The human and animal pathogen Trypanosoma brucei lives extracellularly in the blood of its mammalian host, where it must evade continual surveillance by the immune system whilst obtaining nutrients required for survival. It achieves this through antigenic variation of its major surface protein (the glycosylphosphatidylinositol (GPI)-anchored VSG) and surface compartmentalisation, retaining transporters and receptors essential for the uptake of nutrient in a specialised membrane invagination at the base of its flagellum – the flagellar pocket (the sole site of endocytosis and secretion in the parasite). This PhD exploits a high-confidence, validated cell surface proteome (‘surfeome’) for bloodstream-form T. brucei to test hypotheses about GPI-anchored protein sorting and flagellar pocket retention. It also attempts to contribute towards early-stage development of strategies for disease control through the recombinant production of surfeome components for testing as vaccine candidates.

It has been proposed that sorting of trypanosome surface proteins to their target membrane domain is influenced by protein abundance, glycosylation, or the number of GPI anchors attached (‘GPI valence’). However, none of these hypotheses is sufficient to explain what we now know about the parasite cell surface. Instead, this project tests if the information required to direct GPI-anchored protein sorting is intrinsic to the GPI-insertion signal sequence itself. The GPI signal sequences from five T. brucei surface proteins (that localise to different domains on the parasite surface) were fused to exogenous fluorescent reporter proteins. These signal sequences allowed correct GPI attachment, but did not result in the differential localisations of the respective endogenous proteins, with all fusions diffused across the entire cell surface membrane. Significantly, results presented herein are incompatible with GPI valance being the primary mode of sorting of GPI-anchored proteins, raising further questions as to what controls the delivery of membrane components to the appropriate target membrane domain.

No vaccines exist for protection against African trypanosomiasis. For the development of an effective vaccine, native-like recombinant antigens must be produced and purified. Initial experiments in this PhD project used the commercially available, Leishmania tarentolae-based system LEXSY; but its underperformance led to the development of a novel system based on Crithidia fasciculata (CExSy). A single marker C. fasciculata line (SMC) that expresses the T7 RNA polymerase and the tetracycline repressor protein was generated, along with a suite of plasmids that allowed production of >10 milligrams of GFP per litre of cell culture. Subsequent expression of three invariant, surface-exposed T. brucei antigens enabled characterisation of glycosylation status and isolation of high purity protein. This system may prove useful for downstream biochemical, structural and pre-clinical applications.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Gadelha, Catarina Wickstead, Bill
Keywords:	Trypanosoma; Membrane proteins; Recombinant proteins
Subjects:	Q Science > QL Zoology > QL360 Invertebrates
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	60098
Depositing User:	Miller, Thomas
Date Deposited:	31 Jan 2023 08:22
Last Modified:	31 Jan 2023 08:23
URI:	https://eprints.nottingham.ac.uk/id/eprint/60098

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