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A dissertation submitted to the faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Human African Trypanosomiasis (HAT) is transmitted by Glossina species and remains a serious health problem in Africa. Many aspects of control of the disease have been implemented throughout the years but vector control of tsetse flies has proven to be the most efficient long-term solution. Vector control interventions have been implemented for many tsetse species but relatively little is known about the behaviour of the riverine species, Glossina fuscipes fuscipes. Increased knowledge of this species would improve vector control interventions. This study aimed at: i) understanding the behaviour of tsetse flies around visual devices and odour baits; ii) understanding the behaviour of the flies with regard to human activities; iii) understanding the interaction between the nutritional status of tsetse flies and their attraction to various trapping devices (biconical traps and electric nets); and iv) establishing an age determination curve for field-caught flies. Results showed that visual targets were better attractants then odour-based ones and electric nets performed better than biconical traps. The sticky traps caught 10x more flies (males) than the stationary biconical traps. Sticky traps caught more young flies than the biconical traps which caught more old flies. An age curve was established for flies ranging from 1 day to 60 days old and the fluorescence-based age determination technique, using pteridine levels, has been shown to work for this species. Understanding the behaviour of tsetse flies around trapping devices should lead to improved trapping efficiency. The data gathered will be of importance in assisting with designing and running the Lake Victoria region control operations planned by PATTEC and it will have application in G. f. fuscipes endemic regions in other parts of Africa.

The parasitic pathogens of genus Trypanosoma cause significant morbidity and mortality worldwide. The most well studied Trypanosoma related diseases are African sleeping sickness (Trypanosoma brucei) and African Animal Trypanosomiasis (Trypanosoma congolense). Despite more than 100 years of research these diseases continue to have a devastating impact on the socioeconomic development of Africa. A major impediment to controlling outbreaks is the lack of an effective vaccine due, in part, to the parasite’s ability to continually alter its protein coat while in the host, which results in effectively evading the host immune system. Recent studies have identified Trypanosoma congolense proteins that are selectively expressed during transmission in the tsetse arthropod vector where the parasite’s protein coat is not constantly recycled. Of these proteins, Congolense Insect Stage Specific Antigen (TcCISSA) and Congolense Epimastigote Specific Protein (TcCESP) were selected for characterization based on cellular localization, expression levels and predicted roles in facilitating transmission by the tsetse fly. The goal of the present study is to understand the crosstalk between T. congolense and its vector, the tsetse fly. Revealing the structure of proteins is a crucial step in determining their functions. In order to gain insight into the molecular basis of structure and function of TcCESP and TcCISSA we took various biophysical and biochemical approaches. TcCISSA was recombinantly produced in E. coli, crystallized and diffraction quality data collected to 2.5 Å resolution. Structure determination, however, has been problematic due to the absence of homologous models and the inability to take advantage of SelMet phasing due to the presence of only a single methionine in the sequence. Structure determination efforts are ongoing using multiple approaches including NMR. In contrast to TcCISSA, the size and complexity of TcCESP required insect cells for efficient recombinant production. While crystallization trials have yet to yield diffraction quality crystals, a combination of homology modeling validated by chemical crosslinking and mass spectrometry, and circular dichroism spectroscopy have yielded intriguing insight into the architecture of CESP. Characterizing the function of these proteins offers the potential for rare insight into the molecular crosstalk between the parasite and vector and may support the development of novel transmission blocking vaccines. / Graduate

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The surface molecules (procyclins) of procyclic forms of African trypanosomes (Trypanosoma brucei spp.) are complex mixtures of lipid-anchored glycoconjugates. The procyclins are expressed differentially during the parasite life cycle within the tsetse fly vector. It has been hypothesised that these surface molecules are involved in interactions with molecules of the tsetse fly and may influence differentiation, cell death and tissue tropism. To understand procyclin functions it is necessary to identify and characterise them. This thesis presents a study of the biochemical and immunochemical characteristics of the major surface molecules of Trypanosoma simiae and Trypanosoma congolense, animal pathogens of the subgenus Nannomonas that share the same developmental cycle and tropism within the tsetse vector. Organic solvent extraction, reverse-phase high performance liquid chromatography and enzyme-linked imnunosorbent assay using surface binding monoclonal antibodies were used to isolate membrane molecules of procyclic culture forms (PCF) of both trypanosome species. Gel electrophoresis of the purified molecules revealed two predominant molecular species from each parasite that were broadly similar yet showed different apparent molecular masses and staining characteristics. The molecules were shown to be glycosylphosphatidylinositol-lipid anchored glycoconjugates, comprised mainly of carbohydrates. Each moiety displayed surface-disposed carbohydrate epitopes that were recognised on the surface of both species of trypanosomes by monoclonal antibodies specific for procyclic parasites of the subgenus Nannomonas. The epitopes were previously shown to be displayed on the glutamic acid-alanine rich protein (GARP) of T. congolense, yet neither this protein (as detected either immunologically or by mass spectrometry) nor its encoding gene (as detected by Southern blot analysis) was present in T. simiae. The results indicate that although T. congolense and T. simiae share common carbohydrate surface epitopes, these are displayed on biochemically different molecules. I hypothesise that the surface disposed carbohydrate structures and not the polypeptide moieties are involved in parasite-tsetse interactions since these species have the same developmental cycles in the insect vector. In an attempt to obtain primary sequence information for the T. simiae PCF surface molecules, I identified and characterised an unique open reading frame. This was shown to be expressed as a protein in PCF and is likely a membrane-associated molecule of the subgenus Nannomonas. / Graduate

The problem of controlling tsetse flies in Africa is an old one. The tsetse fly transmits the trypanosome parasites which cause sleeping sickness in humans and disease in cattle. Because cattle are a favoured food source for tsetse much work has been done looking at the use of insecticide treated cattle as a control strategy for the tsetse fly. Such treatment methods possess many advantages; they are safe and relatively environmentally benign, they can be applied by individual farmers without the need for logistically demanding and costly traditional control programmes and, in addition to tsetse flies the insecticides are effective against a wide range of other harmful cattle parasites. The cost of the insecticide is however a significant constraint to the number of livestock keepers who can afford to employ the technique and as a result many cattle remain untreated. Following the discovery that tsetse had a significant predilection for feeding on the legs and belly of cattle, it was hypothesised that restricting the insecticide to only those areas could offer comparable protection to treating the whole animal. Such an approach would use up to 80% less drug and thus make the treatment per animal much cheaper. In addition, preferentially targeting areas favoured by tsetse, and leaving the rest of the animal untreated, preserves some important ecological balances between cattle and their parasites which traditional treatment methods destabilise. This thesis describes the design, implementation and analysis of a longitudinal study run over 8 months in south east Uganda that sought to compare the effect of applying insecticide to cattle only on the regions favoured by tsetse flies. Cattle were recruited to the study and assigned one of four treatment groups; a whole body application of deltamethrin insecticide pour-on; a restricted application of deltamethrin spray, applied to the front legs, ears and belly; a prophylactic trypanocide injection of isometamidium chloride, and a control group, that received no further treatments. All animals in the study were however cleared using twin doses of a trypanocide diminazene aceturate at the start of the study.

Vector-borne diseases such as malaria, leishmaniasis, and African trypanosomiasis are a major scourge to humans and animals in some of the most impoverished nations across the globe. Enabling the transmission of these disease-causing pathogens is a highly sophisticated molecular arsenal of surface proteins. My research focuses on biophysical characterization of these proteins with the ultimate goal of deciphering the molecular crosstalk between pathogen and vector. In support of this goal, I have selected the tsetse fly-transmitted parasites of the genus Trypanosoma, the etiological agent of African sleeping sickness, as a model system. Towards elucidating the molecular mechanism of transmission, I have attempted to characterize structurally three novel proteins; TbFam50.360, TbPSSA2, and TcCISSA and get insight into their functions. Before this study, GARP (Glutamic Acid Rich Protein from T. congolense), and VSG (Variant Surface Glycoprotein from T. brucei) were the only proteins to be structurally characterized in the vector stages of the parasite. Our structural analysis revealed that while the N- terminal region of TbFam50.360 adopted a three-helical structure similar to previously characterized trypanosome surface proteins, ectodomains of both TbPSSA2 and TcCISSA adopted a previously uncharacterized bilobed architecture. The structural analysis further identified putative ligand binding regions in TbFam50.360 and TcCISSA. However, in the absence of binding partners, the exact function of these proteins could not be established. Our lab in conjunction with our collaborators is investigating the binding partners of these proteins within the tsetse. The structures of TbFam50.360, TbPSSA2, and TcCISSA can be added to the repertoire of structurally characterized surface proteins expressed by trypanosomes. The information gained from these first structures of trypanosome surface proteins offer insight into their role in the trypanosome life cycle, and may, in the future, contribute to the control of African trypanosomiasis. / Graduate