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dc.contributor.advisorCoetzer, Theresa Helen Taillefer.
dc.creatorEyssen, Lauren Elizabeth-Ann.
dc.date.accessioned2019-05-31T09:59:07Z
dc.date.available2019-05-31T09:59:07Z
dc.date.created2018
dc.date.issued2018
dc.identifier.urihttps://researchspace.ukzn.ac.za/handle/10413/16300
dc.descriptionDoctor of Philosophy in Biochemistry. University of KwaZulu-Natal, Pietermaritzburg, 2018.en_US
dc.description.abstractAfrican trypanosomosis (AT) is a major obstacle in the establishment of agriculture and economic sustainability in Africa. Animal AT is responsible for large numbers of livestock succumbing to the tsetse transmitted kinetoplastid parasites, Trypanosoma congolense and T. vivax, and as a result, losses in further downstream sectors are experienced. Due to the ability of the trypanosomal parasites to undergo antigenic variation, vaccine candidates are highly unlikely. Peptidases have been identified as virulence factors and are the focus of the development of novel chemotherapies and diagnostics. The metacaspases (MCAs) are a prime example of a chemotherapeutic target and oligopeptidase B (OPB), that of a diagnostic target. Towards the validation of a chemotherapeutic target, recombinant expression was used to obtain an active peptidase which could be enzymatically characterised. Various inhibitors were investigated and their effect on the parasite, analysed. Current diagnostics are based on antibody detection, but an antigen detection format would be preferable as it could differentiate between active and cured infections as anti-trypanosomal antibodies can persist for years. Given the rural, resource-poor locations in the areas of AT incidence, an ideal rapid diagnostic test (RDT) would be robust, affordable, sensitive and specific and requiring minimal training, such as a dipstick test. The MCAs are cysteine peptidases which are found in all kingdoms other than the metazoa, and share a secondary structure fold and catalytic dyad with the metazoan caspases. Since the caspases play a role in apoptosis, it is thought that the MCAs may function in a similar manner. The single copy MCAs of Trypanosoma spp. and Leishmania spp. differ from the multicopy MCAs in that they possess a Pro-, Gln-, Tyr-rich C-terminal domain which is thought to mediate protein-protein interactions. The activity of the single copy MCAs from T. cruzi and L. major has been implicated in the cell cycle of the kinetoplastid parasite. The aim of the project was to express, purify and enzymatically characterise the recombinant and native MCA5 from T. congolense and T. vivax. Using the 3D structures, solved by X-ray diffraction, of MCA2 from T. b. brucei, molecular docking studies were used to validate the inhibition potential of a published library of inhibitors, designed based on the, then, hypothetical structure of TbbMCA2. Since the elucidation of the 3D structure of TbbMCA2 by X-ray diffraction, the inhibitory power of the library of inhibitors against TbbMCA2 and the MCA5s was investigated. The serine peptidase, OPB, has been shown to be released into the host bloodstream by dead and dying parasites. The use of phage displayed scFv (single chain fragment variable) antibodies for the detection of OPB in serum from infected cattle is reported, towards the development of a RDT. Recombinantly expressed TcoMCA5 was shown to autoprocess and over autoprocess when purified using nickel affinity chromatography. Mutagenesis of the catalytic dyad residues reduced the over autoprocessing and the mutated form was enzymatically active at a pH between 6 and 9. This active mutant and purified TcoMCA5 showed a prefence for Arg over Lys at the P1 substrate position and were able to hydrolyse gelatin. Possible novel inhibitors of TbbMCA2 and the MCA5s of T. congolense and T. vivax were identified using a library of ligands (Berg library) based on the P1 specificity of TbbMCA2 and molecular docking. Commercial fluorogenic peptide substrates and inhibitors reported in literature for the characterisation of various MCAs, revealed interactions with the MCAs which should be taken into consideration when modifying the Berg ligands to achieve higher affinity for the MCAs. The application of scFv antibodies, derived from the Nkuku® phagemid library, for the diagnosis of current AAT infections by the detection of OPB, released in the bloodstream of the infected mammalian host, was investigated. After the successful isolation and production of OPB-specific scFv, MCA-specific scFv antibodies can be pursued using the Nkuku® phagemid library. The resulting OPB-specific scFv identified a conserved peptide between T. congolense and T. vivax and was able to detect native OPB in a western blot format. It was predicted that the scFv interacted with OPB in such a way that it would restrict the hinge motion between the C-terminal catalytic and N-terminal regulatory domains of the enzyme and limit access to the active site pocket. The ability of scFv and rabbit-anti-OPB polyclonal antibody in an antigen detection ELISA with sera from T. congolense infected cattle indicated that detection of OPB fluctuated with parasitaemia.en_US
dc.language.isoenen_US
dc.subject.otherTrypanosomosis.en_US
dc.subject.otherDiagnostics.en_US
dc.subject.otherscFv.en_US
dc.subject.otherMetacaspase.en_US
dc.subject.otherOligopeptidase B.en_US
dc.titleMolecular characterisation of metacaspase 5 and the production of oligopeptidase b-specific single chain variable fragment antibodies for potential animal African trypanosomosis chemotherapies and diagnostics.en_US
dc.typeThesisen_US


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