Thesis (M.S.)--Villanova University, 2008. / Chemistry Dept. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Univ., 2009. / Härtill 4 uppsatser.

Thesis (M.S)--Villanova University, 2010. / Chemistry Dept. Includes bibliographical references.

Thesis (MSc (Veterinary Science))--University of Pretoria, 2005. / Includes bibliographical references.

Proefschrift Rijksuniversiteit Groningen. / Met lit. opg. en een samenvatting in het Nederlands.

Procyclic culture forms of Trypanosoma brucei species and antibodies to these parasites were used in developing antibody-detection and antigen-detection assays for diagnosis of African human sleeping sickness. An agglutination assay using live procyclic trypanosomes--the Procyclic Agglutination Trypanosomiasis Test (PATT) was developed for detecting anti-trypanosome antibodies in the sera of trypanosome-infected vervet monkeys and humans. Antibodies to procyclic surface antigens were detected by the PATT in sera of vervet monkeys as early as 7 days post-infection with T. b. rhodesiense. Positive agglutination titres were obtained with sera from monkeys with active, untreated infections and with sera taken soon after successful drug cure. Similar positive agglutination results were also observed using the PATT with sera from T. b. gambiense-infected patients from Cote d'Ivoire and Sudan and with documented sera from T. b. rhodesiense-infected patients from Kenya. No agglutination reactions were observed with preinfection sera from vervet monkeys, with sera from uninfected Canadians or with sera from Americans working in endemic areas. Together these results confirm the diagnostic value of using procyclic trypanosomes to detect anti-trypanosome antibodies in human African sleeping sickness. A double antibody sandwich ELISA using monoclonal antibodies and polyclonal rabbit antibodies to the surface membrane antigens of procyclic trypanosomes was developed. This assay detected circulating trypanosomal antigens in the sera of trypanosome-infected mice and in the sera from parasite-infected patients. However, limited success was obtained with this sandwich ELISA when tested on a larger repertoire of sera from infected humans. Rabbit antibodies made against whole lysates of T. b. rhodesiense procyclics were then employed in an antigen-trapping sandwich ELISA. The results demonstrated the effectiveness of this sandwich ELISA in revealing the infection status of vervet monkeys or humans infected with either T. b. rhodesiense or T. h. gambiense. Trypanosomal antigens were detected in the sera of parasitologically confirmed monkeys and patients but not in preinfection sera nor in control sera from uninfected North Americans. The PATT and the sandwich ELISA exhibited higher sensitivities than the currently employed diagnostic assay for human sleeping sickness, the Card Agglutination Trypanosomiasis Test (CATT), when tested with sera of parasitologically-confirmed humans. The sandwich ELISA was superior to the antibody-detecting PATT and CATT in monitoring trypanocidal drug-treated patients. The overall sensitivity of the PATT and sandwich ELISA was 94.3% and 97.4% and the specificity was 84.5% and 95.5%, respectively. These results thus confirm the diagnostic value of these tests for the diagnosis of human African sleeping sickness. Identification of diagnostically useful antigens was attempted in order to facilitate the adaptation of these diagnostic assays to a simpler format for field application. Pooled sera obtained from trypanosome-infected patients was used as a probe to detect trypanosome antigens separated by high performance liquid chromatography, immunoaffinity and immunoblotting techniques. Most of the antigens were detected in the higher molecular weight range (>62 Kd). Immunization of mice with the target antigens yielded six trypanosome-specific monoclonal antibodies. In a double antibody sandwich ELISA, these antibodies were successful in trapping circulating parasite antigens in sera from trypanosome-infected mice as early as 3 days post-infection. Some of these antigens have been partially biochemically characterized. Trypanosomal antigens were also detected by these antibodies in the urine of infected mice. The antigen-capture sandwich ELISA using either the selected monoclonal antibodies or the rabbit anti-procyclic whole lysate antibodies gave similar results with sera from trypanosome-infected mice, human sleeping sickness patients and uninfected humans from North America and Kenya. The results showed that these MAbs and their antigens were useful in the diagnosis of African human sleeping sickness. / Graduate

Drug resistance together with difficulties involved in the development of new trypanocides are a major problem in the present control of African trypanosomiasis. DNA based diagnostics for drug resistance would overcome problems in the identification of drug-resistant populations and contribute to effective control measures. However, this requires a detailed knowledge of the mode of action and the mechanisms by which trypanosomes can overcome the toxic effects of trypanocides. In this study, a search for molecular differences between a multidrug-resistant isolate of Trypanosoma brucei brucei, CP 547, and a reference drug-sensitive population, ILTat 1.4, led to the identification of a 6.6 kbp extrachromosomal element in the multidrug-resistant population. In light of the involvement of extrachromosomal elements in drug resistance in Leishmana spp. and cancer cells, the identification of the 6.6 kbp element warranted its characterisation. Several different approaches sere attempted before a sequence which hybridised to the 6.6 kbp element its eventually isolated. This sequence is represented by a 108 bp repeat sequence which forms long arrays of tandem repeats. Since N/a III is the sole restriction enzyme that cuts within the repeat, it has been referred to as an N/a III repeal The repeat is flanked by a 5 bp spacer sequence. However, a unique 5 bp direct repeat flanking two complete, and one partial copy of the N/a III repeat may signify the transposition of these sequences. Hybridisation with the N/a III repeat revealed the presence of 'higher' hybridising elements which also appear to be predominantly composed of long tandem arrays of the N/a Ill repeal Through exploitation of the p01) merase chain reaction using arbitrary primers (AP-PCR), additional sequences were identified which are associated with some of the 6.6 kbp and 'higher' hybridising elements. The 6.6 kbp element and some of the 'higher' hybridising elements display features of circular DNA molecules. The 6.6 kbp element also displays some level of size and sequence heterogeneity within different populations of the same trypanosome isolate. The copy number of the 6.6 kbp element is also not stable and appears to be directly affected by the application of selective drug pressure, but a direct association between the presence of the element and the expression of multidrug resistance could not be determined. The N/a III repeat family represents a newly identified repetitive family specific to members of the Trypanozoon subgenus. This repeat family, representing about 5% of the parasite genome, is dispersed through all size classes of chromosomes, in addition to its presence on the extrachromosomal elements. Transcriptional studies of the N/a III repeats have revealed that their transcription is developmentally regulated, since heterogeneous transcripts ranging from greater than 10 kb to smaller than 300 bp are present in the actively dividing long slender bloodstream and insect stage procyclic forms of the parasite but not nondividing, stumpy bloodstream forms. Lastly, the N/a III repeat lacks an open reading frame and transcripts do not appear to have a spliced leader sequence at the 5' end. Furthermore, there is almost an equal representation of polyadenylatcd and non-polyadenlyated transcripts.

Trypanosoma brucei is the vector of African trypanosomiasis in both domestic animals (nagana) and sleeping sickness in humans (Human African Trypanosomiasis). These protozoan parasites are transmitted by the bite of infected tsetse flies (Glossina sp.). African trypanosome infections cause parasite-induced anorexia (PIA) and cachexia in livestock, experimental animals and in humans, and are of economic, veterinary and medical importance in sub-Saharan Africa. The overall aim of this project was to characterise the phenomenon of inappetence in relation to overall energy budget in African trypanosome infection and to then identify potential causal factors and mechanisms. A mouse model of T.b. brucei infection was established with a reproducible time course for the development of inappetence and bodyweight loss. Following an initial parasitaemic peak on day 6 post-infection, a profound period of inappetence was observed from days 7 to 11, accompanied by a 10% loss of body mass. Metabolisable energy intake was reduced, while assimilation efficiency increased significantly but not enough to compensate for the severe reduction in food intake. During the course of T.b. brucei infection, both total energy expenditure and physical activity were reduced. Although physical activity was markedly declined in both light and dark phases, trypanosome infected mice maintained their circadian rhythm albeit at a lower amplitude, with most of the activity occurring at the start of the dark phase. Resting metabolic rate was unchanged in infection. Plasma concentrations of the inflammatory cytokines, IL-6 and TNF-α were increased in infected mice and were associated with inappetence. Reductions of leptin and insulin concentration corresponded to a loss in fat mass. The hypothalamic control of appetite appeared to be normal with increases in appetite stimulating AgRP, decreases in the appetite inhibiting POMC and MC4R. There has been no previous data published on the control of appetite and energy expenditure in African trypanosome infections thus the data presented here provides a novel insight into the pathophysiology of this serious disease, and may lead to new therapies to manage the clinical and veterinary consequences of trypanosome infection.

African animal trypanosomosis or nagana is a disease in livestock caused by various species of protozoan parasites belonging to the genus Trypanosoma particularly T. congolense, T. vivax and T. b. brucei. Nagana is the most important constraint to livestock and mixed crop-livestock farming in tropical Africa. Trypanosomes undergo part of their developmental life in their insect vector, the tsetse fly and part in their mammalian host. Measures for eradicating the continent of the tsetse fly vector include insecticidal spraying, targeting and trapping. Vaccine development has been hampered by the generation of an inexhaustible collection of variant surface glycoproteins that trypanosomes possess and allow for evasion of the host immune system. Anti-disease vaccines aimed at reducing the symptoms of the disease rather than killing the parasite itself have been demonstrated as an alternative approach. Trypanotolerant cattle are able to protect themselves from the disease-associated symptoms. They are able to mount a better antibody response to the CATL-like cysteine peptidase, TcoCATL, compared to trypanosusceptible breeds. Bovine trypanosomosis, however, continues to be controlled primarily by trypanocidal compounds such as isometamidium chloride, homidium and diaminazene that have been developed more than 50 years ago and consequently drug resistance is widespread. Trypanosomal cysteine peptidases have also been proven to be effective targets for chemotherapeutics. TcrCATL, inhibited by the vinyl sulfone pseudopeptide inhibitor K11777, was effective in curing or alleviating T. cruzi infection in preclinical proof-of-concept studies and has now entered formal preclinical drug development investigation. Understanding enzymatic as well as structural characteristics of pathogenic peptidases is the first step towards successful control of the disease. To date no such characterisation of the major cysteine peptidases from T. vivax has been conducted. Although the major cysteine peptidase from T. vivax, TviCATL, has not been proven as a pathogenic factor yet, its high sequence identity with the pathogenic counterparts such as TcrCATL and TcoCATL hold much speculation for TviCATLs role in pathogenocity. In the present study, native TviCATL was isolated from T. vivax Y486, purified and characterised. TviCATL showed to have a general sensitivity to E-64 and cystatin and has a substrate specificity defined by the S2 pocket. TviCATL exhibited no activity towards the CATB-like substrate, Z-Arg-Arg-AMC but was able to hydrolyse Z-Phe-Arg-AMC, the CATL-like substrate. Leu was preferred in the P2 position and basic and non-bulky hydrophobic residues were accepted in the P1 and P3 positions respectively. Similar findings were reported for TcoCATL. The substrate specificity of TviCATL and TcoCATL does argue for a more restricted specificity compared to TcrCATL. This was based on the Glu333 in TcrCATL substituted with Leu333 in TviCATL and TcoCATL. In the case of TcrCATL, the Glu333 allows for the accommodation of Arg in the P2 position. Like other trypanosomal cysteine peptidases, TviCATL was inhibited by both chloromethyl ketones, Z-Gly-Leu-Phe-CMK and H-D-Val-Phe-Lys-CMK. Determining further structural and functional characteristics as well as whether TviCATL, like the T. congolense homolog, TcoCATL, acts as a pathogenic factor, would be important information to the designing of specific chemotherapeutic agents. To date, TcrCATL and TbrCATL (from T. b. rhodesiense) are the only trypanosomal CATL-like cysteine peptidases been crystallised and their tructures solved. This advantage has allowed for the directed design of synthetic peptidase inhibitors. The crystal structure of TcoCATL will be of major significance to the design of specific chemotherapeutic agents. Furtherrmore, understanding the dimeric conformation of TcoCATL is important for vaccine design as immune responses are likely to recognise the dimer specific epitopes. In the current study, the catalytic domain of TcoCATL and TviCATL, were recombinantly expressed in Pichia pastoris and purified to homogeneity. The T. congolense cysteine peptidase pyroglutamyl peptidase (PGP), also proven to be pathogenic in T. b. brucei, was recombinantly expressed in E. coli BL21 (DE3) cells and also purified to homogeneity. Purified cysteine peptidases along with previously purified TcoCATL dimerisation mutants, TcoCATL (H43W) and TcoCATL (K39F; E44P), possessing mutated residues involved in TcoCATL dimerisation, as well as the mutant proenzyme TcoCATL (C25A), were screened for crystallisation conditions using the Rigaku robotic crystallisation suite. One-dimensional needle-like crystals were found for TcoCATL (K39F; E44P). Optimisation of the TcoCATL (K39F; E44P) crystals were analysed for X-ray diffraction. The poor diffraction pattern prompted further optimisations for better crystal quality, which is presently underway. The crystal structure of TcoCATL, with some of the residues involved in dimerisation mutated, will be pivotal in understanding the dimerisation model. Furthermore, the information about the structure will be valuable for vaccine design and chemotherapeutics development. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei brucei cause nagana in cattle. The variable nature of the parasite surface coat has hindered the development of an effective vaccine. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. Two pathogenic factors released during infection are oligopeptidase B (OPB) and TcoCATL (congopain). TcoCATL, a major lysosomal cysteine peptidase, is a member of the papain family C1 cysteine peptidases. RNA interference (RNAi) was used to down-regulate the expression of TcoCATL in T. congolense IL3000 TRUM183:29-13 parasites in vivo during mouse infections. TcoCATL RNAi was monitored in infected mouse blood by comparing the hydrolysis of Z-Phe-Arg-AMC and parasitaemia between mice in which RNAi was induced and control mice. Mice infected with parasites induced for TcoCATL RNAi had lower parasitaemia when compared to control mice. An attempt was also made at deleting the entire CATL gene array in both T. congolense IL3000 and T. brucei 427 Lister strains. The second pathogenic factor studied, OPB, is a cytosolic trypanosomal peptidase that hydrolyses peptides smaller than 30 amino acid residues, C-terminal to basic residues. In order to evaluate the role that OPB play during disease, RNAi was also applied to knock-down the expression levels of OPB in T. brucei T7T and T. congolense IL3000 TRUM183:29-13 strains (TbOPB and TcoOPB respectively). Oligopeptidase B null mutant strains (Δopb) were also generated in T. brucei brucei Lister 427. An attempt was also made to generate OPB null mutants in T. congolense IL3000 parasites. Western blot analysis of the knock-down experiments using chicken anti-TcoOPB peptide IgY showed that only TbOPB levels were reduced in T. brucei T7T parasites induced for RNAi when compared to TcoOPB RNAi induced cultures. Quantitative assessment of a fourteen day induction experiment for OPB RNAi in T. brucei showed an 87% reduction in TbOPB levels when compared to levels on day one. There was no growth effect observed in T. brucei parasites cultured in vitro and induced for TbOPB RNAi. It was concluded that TbOPB is not necessary for the in vitro survival of T. brucei parasites, thus making the generation of OPB null mutants possible. Δopb T. brucei parasites were successfully generated and grew normally in vitro and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that T. brucei OPB (TbOPB) is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatin gel analysis of Δopb null mutants and wild type strains showed an increase in cysteine peptidase activity. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by knocking out TbOPB, and a significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed. However, western blot analysis did not show any increase of TbPOP protein levels in Δopb parasites, suggesting that either TbOPB is responsible for generating an endogenous inhibitor for TbPOP or that another POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants. This study made a significant contribution to an understanding of the interplay between different trypanosomal peptidases that are important pathogenic factors in trypanosomosis. It highlights the need to simultaneously target several trypanosomal peptidases to develop an effective vaccine or chemotherapeutic agents for African animal trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.