Abstract:

Trypanosoma brucei is a tsetse fly-transmitted protozaon pathogen of the genus Trypanozoon that causes sleeping sickness or Human African Trypanosomiasis (HAT) in man and nagana in animals. HAT is invariably fatal unless treated and causes huge economic losses to the affected communities. Therefore, HAT management is of great importance in improving the livelihoods of such communities. HAT management involves accurate diagnosis and chemotherapy. However, HAT is a neglected disease with respect to treatment as well as diagnostics. In this thesis I set out to address some of the problems related to HAT diagnostics and chemotherapy. This work comprises five (5) chapters. Chapter 1.0 which is the Introduction: gives a general overview on Trypanosoma brucei and HAT. Chapters 2.0, 3.0 and 4.0, comprise the results. Chapter 2.0 is an aspect of HAT diagnostics while chapters 3.0 and 4.0 are aspects of chemotherapy. Chapter 2.0 is Comparative genomics and Serodiagnostic evaluation of Tandem Repeat (TR) Proteins in Trypanosoma brucei. In the absence of laboratory equipement, serology is the main stay for diagnosis of HAT under field conditions. Trypanosoma brucei TR proteins were retrieved and identified in silico and genes of selected proteins with expression profiles in the bloodstream form of the parasite were amplified and expressed in E. coli. The recombinant proteins were purified and evaluated using ELISA. Preliminary results obtained show that the selected proteins cannot be used as antigens for HAT diagnosis since they do not distinguish between sera from HAT patients and that from healthy individuals. Chapter 3.0: Mechanisms of drug resistance in Trypanosoma brucei is divided into three parts i) Combined contribution of TbATl and TbMRPA to drug resistance in Trypanosoma brucei. TbATl is anaminopurine transporter responsible for uptake of trypanocides like melarnophenyl arsenicals, pentamidine, diminazene aceturate, isometamidium, cordycepin and tubercidin, while TbMRPA is an efflux transporter of the ABC type which contributes to melanophenyl arsenical resistance when overexpressed. The aim of this work was to investigate what happens when both loss of TbATl and gain in TbMRPA, coincide in the same cell. Results obtained show that the two transporters independently contribute to arsenical resistance in Trypanosoma brucei (Chapter 3.0a). Part ii (Chapter 3.0b) and iii (Chapter 3.0c), investigate the phenomenon of cross resistance between melanophenyl arsenicals and diamidines in Trypanosoma brucei. Drug resistance to melarsoprol and pentamidine was independently induced in the laboratory strains and the mechanisms involved were investigated. In chapter 3.0b the mechanism for cross resistance is not known since only the melarsoprol-resistant isolate had lost the TbATl. In chapter 3.0c however, cross-resistance is associated with the loss of the High Affinity Pentamidine Transporter (HAPT1) activity. In Chapter 4.0: Comparative genomics of metabolic networks from parasites and host, the aim was to determine how networks shrink and to identify potential drug targets. In this work pathways constituting core metabolism of different organisms were broken down into individual reactions and analysed in the perspective of entire networks. Different properties of the networks were compared between parasites and non-parasites. Two enzymes specific to Tiypanosoma brucei', methionine synthase (EC2.1.1.14) and homocysteine methyltransferase (EC2.1.1.10) were knocked down and knocked out, respectively and the generated clones were grown in methionine-free or methionine-supplemented medium. Results obtained show that network integrity rather than scale-freeness has acted as a selective principle for evolution of parasite metabolism. Experiments on methionine synthase and homocysteine methyltransferase show that Trypanosoma brucei is auxotrophic for methionine and that homocysteine methyltransferase and methionine synthase are potential drug targets. Chapter 5.0 is the overall discussion and outlook of all the work done and written in this thesis.