Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The need for alternative or supplementary treatments due to the global problem of microbial resistance towards conventional antimicrobials may be met by the development of novel drugs based on antimicrobial peptides. The antimicrobial peptides of interest to this study were the tyrocidines, cyclic decapeptides produced by Bacillus aneurinolyticus. Although these antimicrobial peptides were the first natural antibiotic to be discovered though a systematic search for antibacterial compounds, information regarding their bioactivity, structure-activity relationships, determinants of bioactivity and mode of action is limited. The aim of this study was to investigate the antibacterial and antiplasmodial activity, as well as to identify determinants of bioactivity modulation, of the natural tyrocidine library. The study indicated that the tyrocidines exhibit significant activity toward Gram-positive bacteria, notably Listeria monocytogenes, and the intraerythocytic parasite, Plasmodium falciparum. Both the antilisterial and antiplasmodial activity was found to be highly dependent on peptide identity and self-assembly. The antilisterial activity of the tyrocidines was shown to be associated with increased self-assembly within a membrane-like environment, which suggested that formation of lytic complexes within the bacterial membrane may play a crucial role in tyrocidine activity. In contrast to the observations for antilisterial activity, the antiplasmodial activity of the tyrocidines was shown to be associated with reduced self-assembly within a membrane-like environment, which suggested that the antiplasmodial activity of the tyrocidines is mediated by a mechanism other than the formation of lytic complexes within the target cell membrane. In addition to the influence of peptide identity and self-assembly, the bioactivity of the tyrocidines was found to be highly sensitive to environmental conditions, notably the presence of calcium. The antilisterial activity, as well as the mode of action, of the tyrocidines was also found to be highly sensitive to tyrocidine-Ca2+ complexation and the concomitant induction of higher-order structures. Tyrocidine-Ca2+ complexation was shown to greatly enhance antilisterial activity and change the mechanism of action from a predominantly membranolytic to an alternative, non-lytic mode of action. The results of this investigation suggest that the alternative mode of tyrocidine activity may be related to complexation with Ca2+. It is hypothesised that such complexation may either (1) promote tyrocidine-DNA complexation, and thus inhibition of transcription and/or replication; or (2) interfere with Ca2+ homeostasis, and thus influence vital cell functions. Overall, it may be hypothesised that tyrocidine activity and mode of action is modulated by a critical play-off between self-assembly, cation-complexation and membrane-interaction. As these modulators of activity are highly dependent on tyrocidine sequence/structure, the wide variety of tyrocidines found in the natural complex may allow for optimal interaction with and activity toward a variety of microbes. / AFRIKAANSE OPSOMMING: Die universele probleem van mikrobiese weerstand teen konvensionele antimikrobiese middels en die wêreld-wye noodsaaklikheid vir alternatiewe of bykomende behandeling mag deur die ontwikkeling van nuwe middels, gebasseer op antimikrobiese peptiede, vervul word. Die antimikrobiese peptiede van belang tot hierdie studie is die tirosidiene, sikliese dekapeptiede wat deur Bacillus aneurinolyticus geproduseer word. Informasie ten opsigte van die tirosidiene se bioaktiwiteit, struktuur-funksieverwantskap, determinante van bio-aktiwiteit en meganisme van aksie was beperk, alhoewel hierdie peptiede die eerste antimikrobiese peptiede was wat ontdek is deur ‘n sistematiese soektog vir antimikrobiese middels. Die doelwit van hierdie studie was die ondersoek van antibakteriële and antiplasmodiese aktiwiteit, sowel as om die determinante van bio-aktiwiteit modulering van die natuurlike tirosidienbiblioteek te ondersoek. Hierdie studie het getoon dat die tirosidiene merkwaardige aktiwiteit teenoor Gram-positiewe bakterië, in besonder Listeria monocytogenes het, asook teenoor die intra-eritrositiese parasiet, Plasmodium falciparum. Daar is bevind dat beide die antilisteriese en antiplasmodiese aktiwiteite hoogs afhanklik is van peptiedidentiteit en self-verpakking. Daar is gewys dat die antilisteriese aktiwiteit van die tirosidiene geassosieer is met verhoogde self-verpakking in ’n membraanagtige omgewing, wat ’n aanduiding is dat die vorming van litiese komplekse in die bakteriële membraan ’n kritiese rol in tirosidienaktiwiteit speel. Kontrasterend tot die waarnemings van antilisteriese aktiwiteit, is getoon dat die antiplasmodiese aktiwiteit van die tirosidiene geassosieer is met verlaagde self-verpakking in ’n membraanagtige omgewing. Dis ’n aanduiding dat die antiplasmodiese aktiwiteit van die tirosidiene gemediëer word deur ‘n ander meganisme en nie die vorming van litiese komplekse in die teikenselmembraan nie. Bykomend tot die invloed van peptiedidentiteit en self-verpakking, is daar bevind dat die bioaktiwiteit van die tirosidiene hoogs sensitief is vir die omgewing, in besonder die teenwoordigheid van kalsium. Daar is ook bevind dat die antilisteriese aktiwiteit, sowel as die meganisme van aksie, van tirosidiene hoogs sensitief is vir tirosidien-Ca2+ kompleksvorming en die gevolglike induksie van of hoër-orde strukture. Daar is gewys dat tirosidien-Ca2+ kompleksvorming die antilisteriese aktiwiteit drasties verhoog en dat die meganisme van aksie verander van ’n oorwegende membranolitiese meganisme na ’n alternatiewe nie-litiese meganisme van aksie. Die resultate van hierdie ondersoek het aangedui dat die alternatiewe meganisme van aksie van tirosidienaktiwiteit moontlik verband kan hou met kompleksvorming met Ca2+. Die hipotese is dat sodanige kompleksvorming moontlik of (1) tirosidien-DNA komplekvorming aanmoedig, en dus transkripsie en/of replikasie inhibibeer of (2) met Ca2+ homeostase inmeng, en sodoende lewensnoodsaaklike selfunksies beïnvloed. Die algemene hipotese is dat tirosidienaktiwiteit en meganisme van aksie deur ’n kritiese spel tussen self-verpakking, katioonkompleksvorming en membraaninteraksie gemoduleer word. Die wye verskeidenheid van tirosidiene, wat in die natuurlike kompleks gevind word, kan moontlik toelaat vir die optimale interaksie met, en aktiwiteit teenoor ’n verskeidenheid van mikrobes, aangesien die aktiwiteitmoduleerders hoogs afhanklik is van tirosidien struktuur/volgorde.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: A number of relevant brewing industry issues associated with malt quality were examined. These included beer foam quality, premature flocculation of yeast during fermentation and antimicrobial factors in malt. The cause of poor foam at a brewery relative to other similar breweries was identified as being related to the boiling temperature during wort preparation and the associated conformational changes of the abundant foam protein lipid transfer protein 1 (LTPl). The temperature range of 96 to 102°C was revealed to be critical. At the higher temperature the denaturation of LTP 1 was more extensive and its effectiveness as a foam protein was reduced. In addition, it was shown that the prominent role of LTPI with respect to foam was as a lipid binding protein, forming a lipid sink and protecting foam from lipid damage. The occurrence of malt associated premature yeast flocculation (PYF) during fermentation was induced in malt by the addition of extra-cellular fungal enzymes to the malt husk or by micro-malting barley in the presence of fungi. In addition, treating malt husk with commercial xylanase or adding commercial arabinoxylan to the fermentation also impacted on yeast flocculation. It was proposed that a range of molecular weight arabinoxylans formed by the enzymatic breakdown of the major barley husk component (arabinoxylan) resulted in PYF. Antimicrobial activity against brewing yeast (Saccharomyces cerevisiae), other fungi and bacteria was found in barley, malt and malt derived wort trub. Wort trub is the non-specific precipitate of protein, polyphenols and lipids formed during wort boiling and which is, to some extend, carried over in the wort to the fermentation. Antimicrobial activity appeared to increase during malting. The growth of brewery collected yeast was inhibited in the presence of brewery production wort when compared to the same wort filtered to remove the trub. Brewery yeast was found to be more sensitive to inhibition than laboratory propagated yeast of the same strain. Different strains of S. cerevisiae were also found to differ in their sensitivity to inhibition. Investigation revealed that the activity originated from the inside of the barley grain and impacted on yeast sugar uptake. However, there was no direct correlation detected between levels of antimicrobial activity in malt and fermentation performance. At high concentrations the factors were microcidal causing cell lysis. Partial characterisation of an antimicrobial extract from malt revealed the presence of a factor between 5 and 14 kDa, containing a cationic peptide component. The optimum pH stability was ±5 when it was also most cationic. The factor easily and irreversibly lost activity at extreme pH and when exposed to certain reagents but was heat resistant in accordance with its survival in wort trub. Preliminary results showed the presence of LTP1 associated with other peptides in the active cationic fraction from the one malt tested. The occurrence of malt related PYF and malt antimicrobial factors are associated with microbial contamination of the grain. The fungi generating the PYF factors from the barley husk while the barley's defence mechanism generates antimicrobial factors to cope with the pathogenic effect of the fungi. In addition there is a potential link between the foam protein LTP 1 and malt antimicrobial activity as LTP 1 or LTP 1 in association with another component(s) is potentially antimicrobial. / AFRIKAANSE OPSOMMING: 'n Aantal problematiese areas in die broubedryf, wat met mout geassosieer word, is ondersoek, naamlik bierskuimkwaliteit, voortydige flokkulering van gis tydens fermentasie en die invloed van antimikrobiese faktore in mout. Die oorsaak van swak bierskuim by 'n spesifieke brouery relatief tot ander soortgelyke brouerye was geidentifiseer as die moutekstrakkookpunt tydens moutekstrakbereiding. Tydens hierdie proses ondergaan dieskuimprotein, lipiedoordrag proteien 1 (lipid transfer protein 1, LTPI), 'n konformasieverandering. Die temperature tussen 96 to 102°C was kritiek t.o.v. ideale konformasieverandering vir skuimaktiwiteit. Denaturering van LTPI het by hoër temperature plaasgevind wat die skuimproteien se aktiwitiet verminder het. Daar is ook bewys dat LTPI 'n verdere rol in bierskuim speel aangesien dit 'n lipiedbindingsproteien is wat die skuimnegatiewe lipiede verwyder. Die voorkoms van moutgeassosieerde voortydige flokkulering van gis (PYF) tydens fermentasie is op twee maniere in mout geinduseer, naamlik: • deur die toevoeging van ekstrasellulêre swamensieme tot die moutdop • deur mikrovermouting van gars in die teenwoordigheid van swamme. Die behandeling van die moutdop met kommersiele xilanase of die toevoeging van kommersiele arabinoxilaan by fermentasies het ook die flokkulering van gis beinvloed. Die hipotese was dat PYF veroorsaak is deur 'n reeks arabinoxilane met verskillende molekulêre massas wat gevorm het tydens die ensimatiese afbraakproses van die primere moutdopkomponent (arabinoxilaan). Antimikrobiese aktiwiteit teenoor brouersgis (Saccharomyces cerevisiae), ander swamme en bakterie was teenwoordig in gars, mout en moutekstrakpresipitaat. Die presipitaat bestaan uit nie-spesifieke presipitate van proteien, polifenole en lipiede wat gedeeltelik in die gekookte moutekstrak agterbly. Daar is gevind dat antimikrobiese aktiwiteit tydens vermouting toe geneem het. Die groeiproses van brouersgis, gekollekteer by 'n brouery, was geinhibeer deur die teenwoordigheid van brouery-geproduseerde moutekstrak in vergelyking met dieselfde moutekstrak wat gefiltreer was om die presipitaat te verwyder. Die brouersgis was meer sensitief heens inhibisie in vergeleke met dieselfde gisstam wat opgegroei is in die laboratorium. Verskillende S. cerevisiae stamme het ook verskille in sensitiwiteit getoon t.o.v. the antimikrobiese komponente in die moutekstrakte. 'n Verdere ondersoek het getoon dat die oorprong van die inhiberende aktiwiteit die interne dele van die gars is, asook dat dit die gissuikeropname beinvloed. Daar was egter geen direkte verband tussen antimikrobiese aktiwiteit in mout en fermentasie effektiwiteit, soos gemeet onder laboratorium toestande, nie. Hoë konsentrasies van die faktore het egter gelei tot seldood weens sellise. 'n Kationiese peptiedbevattende fraksie tussen 5 en 14 kDa en 'n optimale pH stabliliteit van 5 is gevind deur gedeeltelike karakterisering van 'n antimikrobiese moutekstrak. Die aktiewe fraksie se aktiwiteit is onomkeerbaar vernietig by ekstreme pH en blootstelling aan sekere reagense. Die aktiewe verbinding(s) is egter hittebestand en resultate het getoon dat hierdie aktiwiteit die brouproses oorleef as deel van die moutektrakpresipitaat. Voorlopige resultate van die een mout wat getoets is het die teenwoordigheid van LTP 1 getoon, asook die moontlike assosiasie met ander peptiede of kleiner komponente in die aktiewe kationiese fraksie. Die voorkoms van moutgeassosieerde PYF en antimikrobiese faktore in mout word met die mikrobiologiese kontaminasie van gars verbind. Swamme produseer die PYF faktore vanuit die moutdopkomponente, terwyl die plant weer antimikrobiese faktore produseer as deel van 'n beskermingsmeganisme teen die patogene effek van die swamme. Daar is ook 'n potensieele verwantskap tussen bierskuimproteien LTP 1 en antimikrobiese faktore in mout, aangesien LTPI ofLTPl tesame met 'n ander verbinding(s) moontlik antimikrobies is.

Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

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.

No abstract available. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Infectious bursal disease virus (IBDV) has a tropism for the lymphoid tissue of poultry and infects actively dividing and differentiating B-lymphocytes in the bursa of Fabricius. This results in a high mortality rate and severe immunosuppression. These immunodepressed chickens are highly susceptible to secondary infections and have a reduced capacity to respond to vaccination. The principal method to control IBDV is through extensive vaccination using either attenuated live or inactivated IBDV vaccines. However, in recent years due to the emergence of new virulent strains, risk of reversion to pathogenicity, cost considerations and intervention by maternal antibodies, the effectiveness of these vaccines in the veterinary field is being reduced. An alternative approach to prevent infection is by interfering with the binding of IBDV to its receptor protein on the surface of bursal cells. Hence this study was undertaken on the characterisation of a possible IBDV receptor on bursal membranes. Infectious bursal disease virus was isolated from infected bursal tissue using CsCl density gradient centrifugation and visualised with Tris-Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy. Following purification of double stranded RNA from infected bursal tissue and commercially available live IBDV vaccines, a polymerase chain reaction (PCR)-based diagnostic assay based on sequences from the highly conserved viral protein (VP2) region was performed. The presence of the virus was demonstrated by the amplification of a 150 bp band in 2% agarose and 15% nondenaturing PAGE gels. The correctness of this product was confirmed byrestriction digestion with a specific restriction endonuclease (BamHI) that resulted in the predicted digestion fragments of 93 and 57 bp. Following preparation of bursal membrane proteins from uninfected bursal tissue, using sucrose density gradient centrifugation, isolation of IBDV receptor protein was carried out by immobilising IBDV on a Sepharose 4B chromatography matrix. After affinity purification, two prominent protein bands around 40 kDa were visualised using a silver stained Tris-Tricine SDS-PAGE gel. Previous work in this laboratory identified two possible IBDV receptor proteins on bursal membranes of 32 and 40 kDa. Antibodies against peptide sequences derived from the 32 kDa receptor protein were raised in rabbits in the present study. These anti-IBDV receptor peptide antibodies recognised the affinity purified native 40 kDa IBDV receptor proteins in an enzyme-linked immunosorbent assay (ELISA). However, due to the possible epitope denaturation by the reducing treatment buffer prior to Tris-Tricine SDS-PAGE such as SDS and 2-mercapthethanol or detergent (Na-deoxycholate) used during the affinity purification of the IBDV receptor protein, the anti-IBDV receptor peptide antibody did not recognise the receptor protein on a western blot. An inhibition assay was performed in an ELISA format by coating the 40 kDa IBDV receptor protein to see if the anti-IBDV receptor peptide antibody could inhibit IBDV binding to the receptor. The result showed that the anti-IBDV receptor peptide antibody effectively inhibited the binding of IBDV to the receptor. This result could pave the way for reducing IBDV infection by interfering at the viral attachment stage prior to crossing the bursal cell membrane barrier. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

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.

Because tumours are comprised of "self" cells and antigens, they escape recognition by the immune system, which discriminates between "self" and "non-self". One such antigen is cathepsin B, a lysosomal cysteine proteinase, that has been implicated as one of the proteolytic enzymes involved in tumour invasion and metastasis. Cathepsin B autoantibodies could open possibilities which may be useful in cancer immunotherapy. In this study generation of cathepsin B autoantibodies was attempted by manipulating the immune system into recognising and responding to cathepsin B in complex with a "foreign" protein, bovine serum albumin (BSA). Cathepsin B was isolated from rabbit liver using the three phase partitioning (TPP) method, modified by adding t-butanol in the homogenisation buffer. Isolation of cathepsin Band cathepsin L, using this novel method, minimised the formation of artefacts such as a covalent cathepsin L-stefin B complex and produced higher yields of enzyme. Pure rabbit liver cathepsin B was conjugated to BSA, using glutaraldehyde as coupling agent, and administered intramuscularly into rabbits. Another three inoculation protocols, which functioned as controls were: i) free cathepsin B administered intramuscularly, ii) complexed cathepsin B administered intravenously, and iii) free cathepsin B administered intravenously. IgGs isolated from inoculated rabbits' serum were assayed by a three layer ELISA system, immunoinhibition assays and dot blots. The anti-complex (intramuscular) antibodies showed the highest recognition for cathepsin B and were the only antibodies that were immunoinhibitory. This suggests that the immune system was, to some extend, successfully manipulated into recognising the complexed "self" cathepsin B. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

Trypanosoma congolense is a major causative agent of the bovine disease trypanosomosis which has a considerable economic impact on sub-Saharan Africa. Current control methods for trypanosomosis are unsatisfactory and vaccine development has been hampered by antigenic variation. An anti-disease vaccine is based on the idea that disease is caused by the pathogenic factors released by the parasite, rather than by the parasite itself. Therefore, if these pathogenic factors could be neutralised by antibodies produced by vaccination, the disease could be circumvented. The method used here for identification of novel pathogenic factors is based on the concept that trypanotolerant cattle are able to mitigate the disease by generating a specific immune response against a few key antigens (pathogenic factors). Two immuno-affinity columns were therefore prepared: one containing IgG from noninfected sera and a second column containing IgG from trypanotolerant N’Dama cattle serially infected with T. congolense. The differential binding of antigens to the two columns allowed identification of antigens specifically recognised by the immune system of a trypanotolerant animal, i.e. potential pathogenic factors. The most promising antigens identified included several variant cathepsin L-like cysteine peptidases (CPs) and the Family M1 Clan MA aminopeptidases (APs). For the CPs, a study of the genetic organisation was conducted in order to further understand the variability present in this gene family. To this end, two different mini-libraries of cathepsin L-like genes were prepared: one in which genes as different as possible from congopain (the major CP of T. congolense) were selected, and a second which contained all possible genes present in the congopain array. Analysis of the sequences obtained in these two mini-libraries showed that there was significant variability of the genes within the congopain array. Two variants of CPs, chosen for differences in their catalytic triads, were cloned for expression. The recombinantly expressed CP variants differed in substrate preferences from one another and from C2 (the recombinant truncated form of congopain), and surprisingly, all enzymes were active at physiological pH. The two APs were cloned and expressed as insoluble inclusion bodies in an E. coli system, and subsequently refolded. The refolded APs showed a substrate preference for H-Ala-AMC, an optimum pH of 8.0, localisation to the cytoplasm and inhibition by puromycin. The two APs were not developmentally regulated and present in procyclic, metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro. Immunisation of BALB/c mice with the APs did not provide protection when challenged with T. congolense. For an anti-disease vaccine to be protective, it would possibly have to include all pathogenic factors, including the two APs and at least one CP described in the present study. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.