Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Trypanosomes are hemoflagellated protozoan parasites causing chagas disease in South America, Leishmaniasis throughout the world, and African sleeping sickness in humans and nagana in animals in Africa. About 55 million people and 25 million cattle have been estimated to be at risk of contracting African sleeping sickness or nagana respectively. Once injected into the blood stream via the bite of a tsetse fly, the parasite evades the host's immune response by repeatedly changing its surface antigens, thus making the development of a vaccine seem impossible. Furthermore, chemotherapy existing today can be toxic, suggesting that novel methods to prevent diseases caused by trypanosomes are essential. All parasites of the Trypanosomatidae family contain unique microtubular structures called the subpellicular microtubules. Microtubules are made of tubulin and of microtubule associated proteins (MAPs). Unlike other microtubules, the subpellicular microtubules are crosslinked to one another and to the plasma membrane. The unique structure of the subpellicular microtubules has been attributed to unique trypanosome subpellicular MAPs which stabilize the microtubule polymers and crosslink them to one another. Three unique types of subpellicular MAPs have been identified: MARP, which is a high molecular mass MAP that stabilizes microtubules, p52 that is a 52kDa MAP which crosslinks microtubules, and pI5, which is a I5kDa protein which bundles microtubules. Because trypanosome MAPs have been shown to be unique to these parasites, these molecules could serve as useful target sites for therapy. In this study pI5 was cloned and sequenced and shown to contain highly organized, nearly identical tandem repeats with a periodicity of 10 amino acids, rich in positively charged and in hydrophobic amino acids. It was shown that pI5 can also bind phospholipids, suggesting that it may not only bundle the microtubule polymer through its positively charged amino acids but may also crosslink the microtubules to the plasma membrane through its hydrophobic regions, thus contributing to the stable structure of the subpellicular microtubules. To test for the efficiency of pI5 as a vaccine candidate, the recombinant pI5 was cloned into an adenovirus, which was used as a vaccine delivery system for pI5. Mice were vaccinated with the native purified pI5, with the expressed recombinant pI5 and with the adenovirus containing the recombinant pI5 gene (Ad-pI5). The results indicated that pI5 protected 100% of the animals vaccinated with the recombinant molecule (8/8), and 87% of the animals vaccinated with the native protein (7/S), while none of the control animals were protected. Animals that were vaccinated with the Ad-pI5 were protected but so were the control animals vaccinated with an adenovirus containing the lacZ gene. We have shown that vaccination with the adenovirus is associated with an elevated CDS+ T cell response which is known to be trypanostatic (S6), suggesting that animals vaccinated with Ad-pIS may have been protected not only by the specific anti-plS response but also by non specific immunity that was induced by the adenovirus itself. The source of the native and recombinant pI5 was from a different strain of T. brucei that was used for challenge. Since the subpellicular microtubules are common to all members of the Trypanosomatidae family, pI5 may ultimately serve as a common target for therapy to all types of diseases caused by trypanosomes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.