Trypanosoma congolense is a protozoan parasite transmitted by tsetse flies. It causes bovine trypanosomosis, the major disease for livestock in sub-Saharan Africa. Control methods include trypanocidal drugs and vector control, but none is fully satisfactory, due to resistance and environmental issues. A method that would have the greatest impact on controlling the disease is vaccination. However, development of a conventional vaccine has been hampered by the mechanism of antigenic variation, which allows the parasite to evade the host’s immune system. An alternative strategy in vaccine design is to target the bioactive compounds released by dead and dying trypanosomes. This approach is termed ‘‘anti-disease’’, and does not affect the survival of the parasite but targets the pathogenic factors released by the trypanosomes. The development of a successful anti-disease vaccine necessitates knowledge of all pathogenic factors involved in the disease process. Several macromolecules, primarily peptidases, have been implicated in the pathogenesis of trypanosomosis. Pyroglutamyl peptidase type I (PGP) was shown to be involved in abnormal degradation of thyrotropin- and gonadotropin-releasing hormones in rodents infected with T. brucei, but to date no data are available on the T. congolense PGP. Molecular cloning and expression in E. coli of the coding sequence of T. congolense PGP, as well as the enzymatic characterisation of the recombinant protein, are reported here, completed by the development of reverse genetics tools for studies of gene function. A 678 bp PCR fragment covering the complete open reading frame of PGP was cloned and sequenced. The deduced amino acid sequence showed 52% and 29% identity with the T. brucei and Leishmania major enzymes respectively. The catalytic residues Glu, Cys and His described in Bacilus amyloliquefaciens PGP are conserved in the T. congolense sequence. PGP was expressed in bacterial systems as a soluble active, 26 kDa enzyme. The recombinant enzyme showed activity specific for the fluorescent substrate pGlu-AMC, with a kcat/Km of 1.11 s-1μM. PGP showed activity in the pH 6.5-10 range, with maximal activity at pH 9.0. The enzyme was strongly inhibited by sulfhydryl-blocking reagents such as iodoacetic acid and iodoacetamide with a kass of 125 M-1 s-1 and 177 M-1 s-1 respectively. Antibodies raised in chickens against the recombinant enzyme allowed the detection of native PGP in both procyclic and bloodstream T. congolense developmental stages, and displayed complete inhibition of the enzyme in vitro at physiological concentrations. To get insight into the role of PGP in parasite biology and trypanosomosis progression, two types of vectors for reverse genetics studies were developed. For RNA interference, a 400 bp 3′ end segment of the PGP open reading frame was cloned into the plasmid p2T7Ti, that will allow PGP gene down-regulation upon integration into the genome of an engineered tetracycline-inducible strain such as TRUM:29-13. For gene knock-out, several rounds of molecular engineering were carried-out in order to create two plasmid vectors, pGL1184-based (blasticidin resistance) and pGL1217-based (neomycin resistance), each bearing 200 bp-long regions at the 5′ and 3′ ends of the PGP open reading frame. In subsequent studies, taking advantage of the recent advances in culture and transformation of T. congolense, these plasmids will allow the creation of single and double knock-out mutants of PGP. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

The lack of a vaccine candidate due to antigenic variation by trypanosomal parasites, the causative agents of human and animal African trypanosomiasis, requires the disease to be controlled by surveillance, diagnosis and appropriate treatment schedules. Due to the non-specific symptoms along with the toxicity and side effects of the current trypanocides, diagnosis needs to be accurate, cost effective and applicable to active case finding in mostly rural settings. Trypanosomal proteases have been identified as virulence factors as they are essential to the parasites‟ survival. Here the diagnostic potential of previously described virulence factors, oligopeptidase B (OPB), pyroglutamyl peptidase (PGP) and the full length and catalytic domain of the cathepsin L-like peptidases (CATLFL and CATL respectively) from T. congolense (Tc) as well as OPB and CATL from T. vivax (Tv), was determined. These antigens were recombinantly expressed, purified and used to generate antibodies in chickens. The purified recombinant antigens were tested in an inhibition and indirect ELISA format using two separate blinded serum panels consisting of sera from non-infected and experimentally infected cattle, one each for T. congolense and for T. vivax. The tested sera were diluted 1:10 for the TcCATLFL, TcCATL antigens whilst the TvCATL antigen used a 1:100 serum dilution. The TcCATLFL, TcCATL and TvCATL antigens had the highest diagnostic potential in the indirect ELISA format with a 90.91, 92.21% accuracy at the second cut-off and a 77.22% accuracy at the third cut-off along with 0.8084, 0.7785 and 0.8813 area under curve (AUC) values respectively. These antigens show potential for development of lateral flow tests to detect T. congolense and T. vivax infections in cattle. The recently discovered metacaspases (MCAs) have been implicated in caspase-like activity and differentiation in T. b. brucei, T. cruzi and L. major and are considered to be virulence factors. The putative metacaspase 5 gene from T. congolense (TcMCA5) was successfully cloned, expressed within inclusion bodies, resolubilised and refolded using immobilised metal affinity chromatography. Recombinant TcMCA5 was successfully refolded as evident by the hydrolysis of the synthetic peptide substrate, Z-Gly-Gly-Arg-AMC. Autocatalytic processing was observed within the inclusion bodies and the products were purified along with the full length recombinant protein. Anti-TcMCA5 IgY antibodies, raised in chickens, were able to detect the native TcMCA5 along with the autocatalytic processed products within the lysate of the procyclic T. congolense (strain IL 3000) parasites. The diagnostic potential of TcMCA5 still requires verification. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

African trypanosomiasis is caused by protozoan parasites known as trypanosomes, which are transmitted by the tsetse fly, affecting both humans and animals. Trypanosoma congolense is one of the main trypanosome species affecting cattle and causes the disease known as nagana. Control of animal African trypanosomiasis currently relies on chemotherapy and vector control methods, neither of which has proven satisfactory. An effective vaccine against trypanosomiasis would be the most cost effective solution to control the disease; however, due to the phenomenon of antigenic variation, intrinsic to the parasite’s outer coat of variable surface glycoprotein, this has not yet been achieved. Recent vaccine efforts have been centred on identification of invariant parasite antigens for use as vaccine candidates. Trypanosome cytoskeleton components have in recent years been shown to be capable of providing a protective immune response against trypanosome infection. These include tubulin proteins, which form the main components of the cytoskeleton, as well as microtubule associated proteins (MAPs) and paraflagellar rod proteins. In the present study α- and β-tubulin from T. congolense were recombinantly expressed and their immuno-protective potential in mice assessed. Amplification of both α- and β-tubulin ORFs from T. congolense genomic DNA was followed by cloning of the amplicons into the T-vector pTZ57R/T, and thereafter sub-cloning into the bacterial expression vector, pET238a and the yeast expression vector pPICZαA28. Only the α-tubulin amplicon was successfully sub-cloned into pICZAαA28; however, no protein expression was achieved upon transfection of the methylotrophic yeast, Pichia pastoris, with this construct. Subcloning of both α- and β-tubulin inserts into pET28a was successful. Expression of recombinant α- and β-tubulin as fusion proteins with a histidine tag, both at a size of 55 kDa, was achieved in Escherichia coli host BL21 (DE3). Recombinant proteins were successfully purified using nickel chelate chromatography under denaturing conditions. Refolding was first attempted by dilution of purified denatured proteins in a refolding buffer followed by reconcentration, but was largely unsuccessful. A second, more successful refolding method was performed wherein denatured proteins were refolded by application of a decreasing gradient of urea, while bound to a nickel chelate column. Native tubulin from cultured T.congolense procyclics was successfully purified and renatured using a polymerisation/depolymerisation method for use as a control for immunisation. Mice were immunised separately with refolded recombinant α- and β-tubulin, native tubulin or an irrelevant protein VP4AA expressed in the same way as the tubulins. ELISA analysis confirmed the production of antibodies against each protein. Parasitaemia developed in all mice following challenge with T. congolense. Only the group immunised with β-tubulin recorded no deaths during the monitoring period despite the presence of parasitaemia, with 60% of mice immunised with α-tubulin or VP4AA and the no antigen control and no mice from the native tubulin immunised group surviving. The results showed that partial protection against trypanosomiasis caused by T. congolense infection was achieved in the group immunised with β-tubulin and suggest that β-tubulin may have vaccine potential. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The disease causes major losses in revenue in many livestock-producing African countries. The available control methods, including chemotherapeutic drugs and insecticidal spraying, have become environmentally unacceptable. Antigenic variation displayed by the parasites has hindered vaccine development efforts. In this context, rather than focusing solely on the parasite itself, efforts in vaccine development have shifted towards targeting pathogenic factors released by the parasites during infection. Congopain, the major cysteine protease of T. congolense, has been shown to act as a pathogenic factor in the disease process. Analysis of the immune response of trypano-tolerant cattle revealed that these animals have the ability to control congopain activity in vivo. Therefore, congopain is an attractive vaccine candidate. To test the protective potential of congopain, immunisation studies had been conducted in cattle using the baculovirus-expressed catalytic domain of congopain (C2) in RWL, a saponin-based proprietary adjuvant from SmithKline-Beecham. Immunised animals were partially protected against a disease caused by an infection with T.congolense. Unfortunately, subsequent attempts to reproduce these results were disappointing. It was hypothesised that this failure could be due to the different expression system (P. pastoris) used to produce the antigen (C2), or the different adjuvant, ISA206 (Seppic), used, thus hinting towards an epitope presentation problem. Congopain had been shown to dimerise at physiological pH in vitro. Sera from trypano-tolerant cattle preferentially recognised the dimer conformation, advocating for protective epitopes to be dimer associated. For that reason, the present study aimed at improving the antigenicity of congopain through firstly, the elucidation of the protective epitopes associated with the dimer, secondly, the determination of the 3-D structure of the protease in order to map protective epitopes to later design mimotopes, and thirdly improve the delivery of congopain to the immune cells while maintaining the conformation of the protease by using a molecular adjuvant, BiP. A dimerisation model was proposed, identifying the amino acid residues forming the dimerisation motif of congopain. In the present study, particular amino acid residues located in the dimerisation motif were mutated by PCR-based site-directed mutagenesis to generate mutants with different dimerisation capabilities. The congopain mutants were expressed in yeast and their dimerisation capability was assessed by PhastGel® SDS-PAGE. The mutations altered both the electrophoretic mobility of the mutants and their enzymatic characteristics compared to wild-type congopain. This advocated for the involvement of these amino acid residues in the dimerisation process, although they seem not to be the only partakers. Wild-type C2 and mutant forms of C2 were heterologously expressed in P. pastoris and purified to crystallisation purity levels. Crystallisation of these proteins is currently underway, but the results are still unknown. While awaiting the crystallisation results, in silico homology modelling was employed to gain insight into the 3-D structure, using cruzipain crystal structure as a template. The modelled 3-D structure of congopain followed the common framework of cathepsin L-like cysteine proteases. Due to time constraints and awaiting the crystal-derived 3-D structure, the 3-D model of congopain was not exploited to design mimotopes with the potential to provide protection against the disease. As it was shown that protective epitopes are likely to be dimer-specific, maintaining the native conformation of congopain is essential for stimulating a protective immune response in animals. Chemically formulated adjuvants usually contain high salt concentration, at acidic or basic pH, thus might change the conformation of the protease. Adjuvants capable of efficiently delivering the antigen to immune cells while maintaining the conformation of the protease were sought. Proteins belonging to the HSP70 family are natural adjuvants in higher eukaryotes. A protein belonging to the HSP70 family was previously identified in T. congolense lysates and is homologous to mammalian BiP. Congopain was genetically fused with T. congolense BiP in order to improve antigen delivery and production of congopain activity-inhibiting antibodies. The chimeric proteins were successfully expressed in both bacteria and yeasts. The low yields of recombinantly expressed chimeras in yeast and problems associated with renaturation and purification of bacteria-expressed chimeras prevented immunisation studies in mice. However, the groundwork was laid for producing BiP-congopain chimeras for use in an anti-disease vaccine for African trypanosomosis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.