Browsing by Author "Coetzer, Theresa Helen Taillefer."

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Antibody-mediated inhibition of proteases of African trypanosomes.
(2006) Huson, Laura.; Coetzer, Theresa Helen Taillefer.
The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis.
Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors.
(2014) Ndlovu, Sijabulisiwe Faith.; Coetzer, Theresa Helen Taillefer.
African Animal trypanosomosis, also known as Nagana, is a parasitic disease which affects many mammalian species, mainly livestock such as cattle, sheep and goats. The disease also affects humans (Human African Trypanosomosis) and in this case is referred to as sleeping sickness. Nagana is caused by the Trypanosoma parasite, which is transmitted to the host by a bite from the tsetse fly (Glossina spp). The Trypanosoma causing trypanosomosis in animals are Trypanosoma congolense, T. vivax and T. brucei brucei. Vaccine development has been unsuccessful, due to the presence of the variant surface glycoprotein on the surface of parasites which undergoes antigenic variation therefore enabling the parasite to avoid detection by vaccines. A chemotherapeutic drug such as isometamidium chloride combined with diminazene and suramin have also had little success due to the increase in drug resistance. During infection of the host, trypanosomal parasites utilise various proteolytic enzymes such as the oligopeptidases, which hydrolyse important host factors such as peptide hormones. These proteolytic enzymes are thus considered to be pathogenic factors which contribute to the manifestation of various trypanosomosis symptoms such as anaemia, fever, paralysis and disturbances in sleep cycle patterns. It is these pathogenic factors which are now being considered as drug targets in the hope to eradicate the spread or continuous advancement of trypanosomosis. Three trypanosomal pathogenic factors, which are serine oligopeptidases which belong to the prolyl oligopeptidase family of serine proteases (Clan SC in subfamily S9) were the focus of this study, namely, prolyl oligopeptidase (POP) from T. b. brucei (TbPOP) and T. congolense (TcoPOP) as well as oligopeptidase B (OPB) from T. congolense (TcoOPB). The full length TbPOP gene was cloned into pTZ57R/T cloning vector and successfully sub-cloned into pET32a expression vector and recombinantly expressed in its insoluble form at a size of approximately 100 kDa using the Escherichia coli BL21 DE3 expression system. TbPOP expression was confirmed by western blot probed with anti-His tag antibodies. Expression of TbPOP was optimised under varying temperatures and IPTG concentrations in an attempt to solubilise the inclusion bodies. However, the protein was expressed as part of inclusion bodies. Therefore, urea denaturation was used for its solubilisation. Following solubilisation, recombinant TbPOP was partially purified on a Ni2+ affinity resin. Further attempts to purify TbPOP by molecular exclusion chromatography (MEC) were unsuccessful, this could be due to aggregation of the protein during the refolding step. Therefore refolding by a Sephadex G-25 desalting column was attempted as it removes some impurities. However, further purification by MEC and ion exchange chromatography (IEC) were unsuccessful. The full-length TcoPOP gene was successfully cloned into pGEM-T® cloning vector and subsequently sub-cloned into pET32a expression vector. However, upon sequencing of the plasmid DNA, it was discovered that a mutation had occurred in the recombinant TcoPOP DNA sequence forming the stop codon “TAG” which resulted in the termination of protein expression, therefore, further work on TcoPOP was not pursued. TcoOPB was successfully recombinantly expressed in pET28a using the E. coli BL21 DE3 expression system. The protein had a size of approximately 80 kDa. The protein was affinity purified using a Ni2+ affinity resin. Expression of TcoOPB was confirmed by western blot using chicken raised anti-TcoOPB antibodies. Cross-reactivity of chicken anti-TcoOPB antibodies with TbPOP was also assessed and no cross-reactivity was found which was expected as POP and OPB only share 25% sequence identity. In order to determine the biochemical characteristics of TbPOP and TcoOPB, various activity assays and kinetics studies were conducted. It was found that TbPOP was able to hydrolyse type I collagen from rat tail. In contrast however, TbPOP was unable to digest gelatin which is a denatured form of collagen. Upon further analysis of TbPOP with the synthetic peptide substrate Z-Gly-Pro-AMC, it was found not to have activity as it was unable to hydrolyse the substrate, this is thought to be due to the misfolding of the protein during the refolding step. TcoOPB on the other hand was unable to hydrolyse either collagen or gelatin. Further biochemical analysis of TcoOPB was conducted using synthetic peptide substrates, the kinetic parameters of TcoOPB Km, kcat/Km were determined and it was found that OPB had a high affinity for the substrates Z-Arg-Arg-AMC, Z-Gly-Gly-Arg-AMC, H-Ala-Phe-Lys-AMC and Z-Pro-Arg-AMC and lower affinity for the substrates H-Pro-Phe-Arg-AMC, H-D-Val-Leu-Lys-AMC, Z-Gly-Pro-AMC, Suc-Ala-Phe-Lys-AMC,Boc-Leu-Gly-Arg-AMC. OPB was also found to have an optimal pH of 8 – 9 and retained 79% of its optimal activity at the physiological pH of 7.4. TcoOPB was found not to have good diagnostic potential as an indirect ELISA revealed that the antigen was unable to detect antibodies in T. congolense infected cattle sera. This study laid the foundation to conduct further studies on TbPOP, TcoPOP and TcoOPB as chemotherapeutic and diagnostic targets for Nagana.
Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.
(2011) Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection.
Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings.
(2013) Mkhize, Phumzile.; Laing, Mark Delmege.; Coetzer, Theresa Helen Taillefer.
Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea.
Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases.
(2011) Jackson, Laurelle.; Coetzer, Theresa Helen Taillefer.
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.
Enzymatic characterisation of a CATL-like protease from Trypanosoma brucei brucei and small-subunit rRNA sequence based phylogenetic analysis of freshwater fish trypanosomes.
(2021) Mthethwa, Bongumusa Comfort.; Coetzer, Theresa Helen Taillefer.; Willows-Munro, Sandi.
Abstract available in PDF.
Epitope mapping of a trypanosomal cysteine proteinase.
(2003) Mkhize, Pamela Phumelele.; Coetzer, Theresa Helen Taillefer.
Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in Sub-Saharan Africa. Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly responsible for the disease, but rather causes illness through the release of pathogenic factors. One of the major pathogenic factors released by trypanosomes is proteinases. Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that inhibit congopain activity. Congopain thus has vaccine potential. This study describes the mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction program. Peptides selected were from the two forms of congopain called CP1 and CP2. Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2). This suggests that the peptides selected have promise for use in vaccines. The peptides were also used to determine whether they are natural immunogenic epitopes of CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T. congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In order to distinguish between T. congolense and T vivax infection, two different peptides from the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from infected cattle. Although anti-peptide antibodies produced against the two C-terminal extension peptides were specific for their respective peptides, thereby indicating the discriminatory power of the peptides selected, there was cross-reactivity by the sera from T. congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay. In addition to attempting to define the epitopes of congopain, preliminary studies to increase the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic presentation of an entrapped antigen. This study reports the interaction between congopain and alpha 2-macroglobulin. The preliminary results of this study showing congopain-alpha 2-macroglobulin interaction could be used to explore the possibility of increasing the immunogenicity of congopain and congopain epitopes by complexing these to alpha 2-macroglobulin. Congopain epitopes complexed with alpha 2-macroglobulin could be used to form a peptide-based vaccine.
Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis.
(2008) Bizaaré, Lorelle Claire.; Coetzer, Theresa Helen Taillefer.
The protozoan parasite Trypanosoma congolense is one of the aetiological agents of African animal trypanosomiasis that is transmitted by the tsetse fly. The parasite causes nagana in animals and affects livestock throughout sub-Saharan Africa. The toxicity of available drugs and the emergence of drug resistant parasites have affected the treatment of trypanosomiasis. Control of the disease has also been difficult due to ineffective vector control and the potential of trypanosomes to express hundreds of antigenetically distinct proteins on their surface. Vaccination against trypanosomiasis has been thought to be a possible control method. Since a vaccine based on variable surface proteins of the parasite is unlikely, research has been directed towards the identification of invariant pathogenic factors of the parasite as potential targets for therapy. Congopain, the major cysteine protease of T. congolense has been implicated in the pathology of the disease. Antibodies against congopain are known to contribute to the mechanisms of natural resistance to trypanosomiasis known as trypanotolerance by neutralising the pathogenic effects of the enzyme. Oligopeptidase B (OpdB), a trypanosomal serine protease has also been associated as a pathogenic factor of the disease. It is released into the host’s circulation by dead or dying parasites and retains its catalytic activity since it is insensitive to host serum inhibitors. In the present study, the catalytic domain of congopain (C2) and the use of alpha-2-macroglobulin (α2M) as an adjuvant were investigated for their potential use in an anti-disease vaccine. α2-Macroglobulin has been used to varying degrees to target different antigens to cells of the immune system and enhance their immunogenicity. A previous study showed that antibodies raised in rabbits against C2 complexed to α2M gave a higher percentage inhibition than antibodies made using C2 mixed with Freund’s adjuvant. In the present study, goats were immunised with C2 complexed with α2M to confirm the enhanced immunogenicity of C2 and the production of anti-C2 antibodies with superior inhibitory properties. Following immunisation, goats were challenged with T. congolense (strain IL 1180) and showed sustained antibody production during the two month infection period. Goat antibodies made using C2 in complex with α2M inhibited the hydrolysis of hide powder azure by C2 by 96%. Maximum inhibition of the hydrolysis of azocasein was observed to be 63% and hydrolysis of Z-Phe-Arg-AMC by C2 was inhibited by 73%. In order to determine the vaccine potential of OpdB, protein was recombinantly expressed as a glutathione-S-transferase fusion protein in the pGEX expression system and purified by glutathione agarose affinity chromatography and molecular exclusion chromatography. Since a small yield of protein necessitated several rounds of expression and extensive purification, OpdB was subsequently expressed as a His-tagged fusion protein in the pET bacterial expression system. Recombinant protein was easily purified using nickel chelate affinity chromatography. Purified OpdB was used with alum for the immmunisation of mice to produce antibodies capable of inhibiting enzyme activity. Following immunisation, mice were challenged with T. congolense (strain IL 1180) and also showed sustained antibody production following two months infection. Since all mice died, the administration of OpdB conferred no protection; however, anti-OpdB mouse antibodies inhibited 86% of OpdB activity against the substrate Z-Arg-Arg-AMC. In addition immunised mice were observed to survive 40% longer than control mice as they had previously been immunised with OpdB and were able to mount a rapid immune response against this pathogenic factor during infection. In general it could be concluded that immunisation of goats with C2 in complex with α2M produced antibodies with superior inhibitory properties. The immunisation of mice with OpdB and alum also produced inhibitory antibodies and previous administration of OpdB enabled mice to mount a rapid immune response against OpdB during infection. Antibody mediated enzyme inhibition demonstrates the potential use of C2 and OpdB as vaccines that may contribute to the development of an effective anti-disease vaccine.
Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools.
(2012) Mucache, Hermogenes Neves.; Boulangé, Alain François V.; Coetzer, Theresa Helen Taillefer.
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.
Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes.
(2011) Kangethe, Richard Thiga.; Coetzer, Theresa Helen Taillefer.
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.
Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis.
(2013) Baiyegunhi, Omolara Olujimi.; Coetzer, Theresa Helen Taillefer.
Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a need for identifying new diagnostic antigens for the serological diagnosis of trypanosomiasis, a disease of humans and animals in Africa caused by protozoa belonging to the genus Trypanosoma. Invariant surface glycoproteins (ISGs) are present in most strains of the parasite and have the potential to replace the variable surface glycoproteins as diagnostic antigens. In order to avoid the challenges of in vivo culturing of bloodstream form (BSF) trypanosomes in laboratory animals, ISG65 and ISG75, the two most common ISGs were heterologously expressed in Escherichia coli and Pichia pastoris expression systems. The extracellular domains of ISG65 and ISG75 of both T. b. brucei and T. b. gambiense were amplified by PCR from genomic DNA using appropriate primers to give inserts of 1121 bp and 1342 bp sizes. These were sub-cloned into the pGEX-4T1 and pET28a expression vectors. Chemically competent E. coli BL21 (DE3) were transformed using the resultant plasmids and the transformed E. coli cells were used for heterologous protein expression. The expressed proteins were purified by three phase partitioning (TPP), nickel or glutathione affinity and molecular exclusion chromatography and analysed by reducing SDS-PAGE. The glycosylation status of ISG65 and ISG75 expressed in the M5 strain of P. pastoris, which has an engineered N-glycosylation pathway that produces glycosylated proteins similar to what is obtained in trypanosomes, was determined. The enzymatic action of Endoglycosidase H resulted in a shift in the electrophoretic migration of ISG65 but not ISG75 on SDS-PAGE, confirming N-glycosylation. Anti-ISG65 and anti-ISG75 antibodies were produced in chickens and affinity purified using the respective recombinant proteins immobilised on affinity matrices. The antibodies recognised native ISG65 and ISG75 respectively in western blots of lysates of T. b. brucei parasites cultured in vitro. Similar recognition of the native ISGs by the anti-recombinant ISG antibodies was also obtained using immunofluorescence microscopy of fixed T. b. brucei parasites. The results obtained demonstrate the potential of application of the recombinant ISG65 and ISG75 and their respective antibodies in the diagnosis of African trypanosomiasis.
Identification and characterisation of novel pathogenic factors of Trypanosoma congolense.
(2010) Pillay, Davita.; Coetzer, Theresa Helen Taillefer.
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.
Identification of infectious bursal disease virus (IBDV) receptors through the use of recombinant capsid protein, VP2.
(2014) Brien, Kayleen Fransiena.; Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) is a non-enveloped Birnavirus which infects the immature antibody producing B-cells of the bursa of Fabricius in young chickens. The virus causes infectious bursal disease (IBD) which is highly contagious and immunosuppressive. A compromised immune system in infected chickens leaves them susceptible to other opportunistic pathogens and as a result increases their mortality rate. Major economic losses in the commercial poultry industry are subsequently experienced in affected regions. Currently vaccines are used to control IBDV infection, however, their efficacy is affected by factors such as the presence of maternally derived antibodies in young chickens which reduces vaccine load, the continuous emergence of new virulent IBDV strains and bursal atrophy caused by some vaccines. It is therefore important to consider new ways of controlling the virus such as targeting specific stages in the virus life cycle. Since virus attachment to host cell receptor(s) is the most crucial step in the virus life cycle, developing novel antiviral agents which prevent viral entry represents a good alternative strategy for IBDV control. Identification of receptor binding proteins and receptors of host cell membranes is required for antiviral development. The receptor binding protein and outer capsid of IBDV is VP2, however, the receptor(s) utilised by IBDV to gain entry into host cells have not been conclusively identified. Recombinant VP2 was used to identify possible IBDV receptor(s) on bursal plasma membranes using a virus overlay protein binding assay (VOPBA) and affinity chromatography. Therefore, VP2 was heterologously expressed in an Escherichia coli and a Pichia pastoris expression system as a 64 kDa fusion protein and a 47 kDa protein respectively. In addition, both systems expressed VP2 as high molecular mass proteins which were confirmed by electro-elution and western blotting. Although purification of VP2 expressed in the E. coli system was a challenge because VP2 expressed as inclusion bodies, polyclonal chicken anti-VP2 antibodies were produced using VP2 expressed in this system. Purification of VP2 expressed in P. pastoris was easier and produced a greater yield of VP2 which was used to produce a VP2-coupled affinity matrix for the purification of chicken anti-VP2 antibodies and for the purification of VP2-binding proteins of the bursal plasma membrane. Moreover, peptides were selected from the VP2 amino acid sequence and use to raise polyclonal chicken anti-VP2 peptide antibodies for comparative identification against chicken anti-VP2 antibodies of possible IBDV receptor(s). Two IBDV VP2-binding proteins with molecular masses of 70 and 32 kDa of the bursal plasma membrane were identified in a VOPBA using recombinant VP2 or IBDV and chicken anti-VP2 antibodies. In addition to the VOPBA, four IBDV VP2-binding proteins with molecular masses of 70, 60, 45 and 32 kDa were affinity purified on a VP2-coupled affinity matrix. Analysis of the affinity purified proteins by mass spectrometry identified five proteins which share common peptides which include, the Ig-gamma chain and Ig-lambda chain of Gallus gallus, outer major protein of Serratia marcescens, the 60 kDa chaperonin of Pseudomonas fluorescens and elongation factor-Tu of Yersinia pestis. The results strongly suggest that an Ig-receptor like protein may form part of the IBDV receptor, however, much further work is required in order to establish whether the chicken homologues of the identified bacterial sequences are part of the putative bursal receptor. It is believed that the bacterial proteins contain common peptides with chicken proteins of the chicken genome which has not been fully annotated as yet. Taken together, this study successfully used VP2 to identify possible IBDV receptor(s) on bursal plasma membranes which could ultimately lead to the development of antiviral agents targeted at IBDV entry.
Identification of possible infectious bursal disease virus receptors.
(2000) Edwards, Thomas Jonathan.; Coetzer, Theresa Helen Taillefer.; Horner, Roger F.
Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system in chickens. Infection by the virus leads to destruction of the bursa and immunosuppression. Infection by virulent strains may result in mortality. Current methods to combat the virus involve the use of vaccines. These are usually a mixture of live attenuated and oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated antibodies. In addition, the vaccines result in damage to the bursa. Identification of a receptor for IBDV could result in the development of either treatment for the virus or superior vaccines by interfering with the attachment of the virus to host cells. Several methods for identifying IBDV binding proteins from the membranes of cells from the bursa of Fabricius were examined. Affinity chromatography of IBDV binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B allowed separation of a number of virus binding proteins. In contrast, virus overlay protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved less; conclusive. Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These were further examined by N-terminal amino acid sequencing of the whole protein and N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the protein respectively. The 40 kDa protein showed homology with human synovial stimulatory protein involved in the formation of autoantibodies in rheumatoid arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa protein complex appeared to consist primarily of a 40 kDa protein when examined by reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western blots using sera from rheumatoid arthritis patients revealed interactions between several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using serum from one of the five patients showed a strong interaction at approximately 80 kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and antibodies in rheumatoid arthritis sera. The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A section of this sequence was amplified by PCR from chicken DNA and RT-PCR from chicken RNA using degenerate primers constructed from the established N-terminal amino acid sequences and chicken codon usage tables. The fragment produced upon amplification from chicken DNA and RNA did not correspond to the predicted size of 177 bp. In contrast, when the RT-PCR product was heated and snap cooled before examination by agarose gel electrophoresis, the product consisted of two fragments, one of approximately 400 bp in size and one of approximately 200 bp in size. The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible receptors for the virus could allow for the development of vaccines and/or treatment strategies for the virus. Treatment strategies or vaccines would be based on blocking of the interaction between IBDV and chicken host cells. Peptide mimics of the epitopes involved in such interactions could possibly achieve this.
Infectious bursal disease virus receptor identification with anti-peptide antibodies.
(2004) Habte, Habtom Haileselassie.; Coetzer, Theresa Helen Taillefer.
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.
Investigation into the major surface proteases of African trypanosomes.
(2016) Delport, Alexandré Marie Chaplin.; Coetzer, Theresa Helen Taillefer.
The unicellular parasite of the genus Trypanosoma infects a number of mammalian species including livestock and humans. In sub-Saharan Africa three main parasitic species cause disease: Trypanosoma brucei, T. congolense and T. vivax. The lack of sensitive diagnosis and increased drug resistance leaves an avenue in trypanosome research for exploring novel virulence factors as diagnostic and chemotherapeutic agents. The work reported in this dissertation involved investigation of the identified virulence factor, the Major Surface Protease (MSP), of African trypanosomes. The MSPs comprise a group of metalloproteases which have been found in over 13 Leishmanian species, T. cruzi, T. brucei and T. congolense as well as other Kinetoplastids. In this study, putative M8 metalloprotease sequences were also identified in the T. vivax genome. These putative sequences were grouped into four classes of protein, TvMSP-A, -C, -D and -E by phylogenetic comparison with other MSPs. Three-dimensional modelling showed high structural identity with leishmanolysin from Leishmania major. The T. vivax MSP sequences were used, in conjunction with T. brucei and T. congolense sequences, to select immunogenic peptide regions to produce anti-peptide antibodies. Three peptides were selected with the intention to 1) detect both TbMSP-B and TcoMSP-B (peptide Tb/TcoMSP:303-314, cross-species), 2) detect only TbMSP-C (peptide TbMSP:400-412) and 3) detect only TvMSP-C (peptide TvMSP:686-697). They were used to generate two types of detection molecules: complete IgY anti-peptide antibodies and single chained fragments (scFvs), with the capability to detect the peptide in an ELISA format. Single scFv expressing E. coli colonies were successfully selected and shown to detect two (Tb/TcoMSP:303-314 and TbMSP:400-412) of the three peptides. The anti-peptide antibodies, produced in chickens, were used to successfully detect native MSP within T. brucei and T. congolense parasite lysates; however, cross-reactivity between species was seen. The T. brucei MSP-C class of protease was successfully cloned, expressed and purified, although, numerous truncated proteins and gene mutations occurred [truncated (t)TbMSP-C]. The expression constructs rTbMSP-C and rTcoMSP-C were hence synthesised. These two enzymes were successfully expressed and purified and were shown to form high molecular weight multimers. Furthermore, the enzymes were able to cleave the peptide substrate H-Suc-Leu-Tyr-AMC with acidic pH optima and activity was inhibited with metalloprotease inhibitors, EDTA and 1,10 phenanthroline. The successful detection of rTcoMSP-C by T. congolense infected cattle sera was also observed. tTbMSP-C, rTbMSP-C and rTcoMSP-C were detected with the chicken anti-peptide antibodies and it was again found that these antibodies cross-reacted with different species MSPs. The high identity shared between MSPs from all Trypanosoma species made selecting species-specific antibodies difficult. Further work to detect native MSP-C protease within infected sera or blood would give a definitive conclusion of its use in diagnostics. Moreover, antibodies that detect just T. brucei and T. congolense still need to be produced. Preliminary activity assays were performed on rTbMSP-C and rTcoMSP-C but, additional research on the kinetics of these proteases is still needed. In summary, it was shown that MSPs have the potential to be novel diagnostic markers especially for cross-trypanosomal species detection. Furthermore, activity of rTbMSP-C and rTcoMSP-C has substrate cleavage specificity and pH optima that are comparable with leishmanolysin.
Investigation of the interaction between antiretroviral drugs and the mucosal microbiome in African women.
(2022) Sibeko, Nomacusi Sibonganjalo Lindiwe.; Gumbi, Pamela Phumelele.; Coetzer, Theresa Helen Taillefer.
Abstract available in PDF.
Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen.
(2011) Hadebe, Sabelo Goodman.; Boulangé, Alain François V.; Coetzer, Theresa Helen Taillefer.
African animal trypanosomiasis is a major threat to African agriculture causing a loss estimated to 4.5 billion US$ per annum. Trypanosoma congolense is the major causative agent in African animal trypanosomiasis and is transmitted by tsetse flies of the Glossina spp. Congopain, a major cathepsin L-like cysteine peptidase in T. congolense is associated with trypanotolerance in N‘Dama cattle and is a target for an anti-disease vaccine. It is suggested that trypanotolerant cattle control the disease by antibody mediated neutralisation of congopain, and that immunisation of cattle against congopain can mimic trypanotolerance resulting in minimised disease pathology. Susceptible cattle immunised with recombinant catalytic domain of congopain, C2, produced high levels of anti-congopain IgG specific antibodies against congopain, maintained weight and exhibited less severe anaemia. However, there was no effect on the establishment of T. congolense infection and acute anaemia development in trypanosusceptible cattle. It has been suggested that failure of congopain to give full protection of the host may be due to poor presentation to the immune system by conventional adjuvants used in previous studies. The aim of the present study was to improve the presentation of the catalytic domain of congopain (C2) to the immune system, by linking it to the proposed molecular adjuvant, BiP, an ER localised HSP70. A further aim was to localise the domain(s) of BiP where the adjuvant properties reside. BiP consists of an ATPase domain (ATPD), a peptide binding domain (PBD) and a C-terminal domain (C-term). Consequently, BiP69, BiP69 lacking the C-terminal domain (BiP60), BiP coding fragments (ATPD, PBD and C-term) and the C2 coding sequence were amplified by PCR from either genomic T. congolense DNA or plasmid DNA. The PCR products were each sub-cloned into a pTZ57RT vector, and C2 cloned into a pET-28a expression vector. The BiP coding fragments were inserted into the recombinant pET-28a-C2 vector, resulting in pET-28a-BiP69-C2, pET-28a-BiP60-C2, pET-28a-ATPD-C2, pET-28a-PBD-C2 and pET-28a-C-term-C2 coding chimeras. The fusion proteins were expressed in an E. coli system as insoluble inclusion bodies at the expected sizes of 96 kDa (BiP69-C2), 88 kDa (BiP60-C2), 47 kDa (PBD-C2), 34 kDa (C-term-C2) and 27 kDa (C2). However, the ATPD-C2 fusion protein was expressed at a larger and smaller size in different attempts. Protein expression was confirmed by western blots using anti-BiP antibodies and anti-congopain N-terminal peptide antibodies. Recombinantly expressed peptide binding domain (PBD)-C2, C-terminus-C2, BiP69-C2, BiP60-C2 chimeras and a BiP69 fusion protein were purified and refolded by a Ni-NTA based one-step on-column refolding method. Bacterial proteins co-purifying with BiP69-C2 and BiP60-C2 chimeras were removed by incubation with 5 mM ATP in the dissociation buffer, but poor yields resulted in using these chimeras as non-pure proteins. Immunisation of Balb/c mice with the BiP69-C2 fusion protein chimera induced a higher antibody response to C2 compared to immunisation with the BiP69/C2 mixture or with C2 in Adjuphos/Quil A. BiP69-C2 and PBD-C2 chimeras and BiP69/C2 mixture induced a robust antibody response to BiP69, but no correlation could be made with the contribution to control of parasitemia and disease induced pathology. Mice immunised with BiP69-C2 and PBD-C2 chimeras showed a better booster effect of T. congolense infection with higher anti-C2 antibody stimulation compared to control groups. Immunisation did not change the establishment of T. congolense infection and anaemia development in most immunised groups. However, mice immunised with the BiP69/C2 mixture and with the PBD-C2 chimera produced anti-C2 antibodies possible contributing to clearing parasites 10 days and 16 days earlier respectively, than mice immunised with BiP69-C2, C-term-C2 and BiP60-C2 chimeras and PBS, C2 and C2 in Adjuphos/Quil A control groups and showed no clinical symptoms of the disease. There was no significant difference in percentage mice survival between BiP-C2 chimera immunised mice and control groups immunised with C2 alone or with a mixture of Adjuphos/Quil A or immunised with PBS. In the present study, it was shown that BiP69 has adjuvant effects when linked to C2 and that its peptide binding domain acts as an adjuvant. It is possible that the removal of the C-terminal domain reduced the adjuvant potency of the peptide binding domain suggesting a prominent role in the adjuvant effect of the BiP molecule. Finding the exact role of the C-terminal domain in the adjuvant effect of BiP would be of utmost interest, and would involve comparing anti-C2 antibody response produced by immunisation with C2 linked to the peptide binding domain with or without the C-terminal domain. Future work includes repeating this study in trypanosusceptible cattle to confirm these findings.
Methods for serological and PCR detection of Salmonella enteritidis in chickens.
(2003) Meyer, Brendan.; Coetzer, Theresa Helen Taillefer.; Horner, Roger F.
Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently one of the leading causes of human food poisoning in the world. It is believed that contaminated poultry products, especially eggs and egg products, have been responsible for the dramatic increase in the incidence of this Salmonella serotype. Detection of S. entertidis has conventionally involved bacteriological examination of samples, yet these procedures are time-consuming which could lead to the rapid spread of S. enteritidis through commercial flocks and potentially cause a human health risk. A number of alternative detection techniques, mostly based on serological methods, have been reported as effective diagnostic assays. However, some of these reports have not been supported by representations of SDS-PAGE gels or Western blots. The objective of this study was the evaluation of these serological techniques as well as a PCR amplification technique, which has been reported to show promising results as a diagnostic method. The techniques discussed in these reports were evaluated with regards to how rapid they were, their specificity and their potential for use in local diagnostic laboratories. Antigens from the outer surface of S. enteritidis were purified by several methods and their antigenicity was tested by separating the antigens by means of SDS-PAGE, followed by Western blotting using sera of chickens infected with S. enteritidis. A high degree of cross reactivity was observed with many of the antigens tested, especially the lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) which had previously been reported as containing antigens which could be used for specific detection of S. enteritidis. This cross-reactivity could be explained by the conserved nature of many of the LPS and OMP antigens among the Salmonella serotypes tested. A fimbrial antigen, SEF14, which has been reported as a novel antigen, was seen as a prominent band at 14.3 kDa and was found to react with antibodies against S. enteritidis, yet not to the specificity levels described in previous reports. PCR amplification of the sefA gene sequence, which encodes for the SEF14 fimbrial antigen, was found to give a predicted product of 310 bp when using a previously described oligonucleotide primer pair. This amplified product was found to be specific for S. enteritidis and other serogroup D Salmonella serotypes that are not poultry pathogens The cross-reactivity observed with many of the serological techniques used in this study, meant that detection of S. enteritidis infection in chickens was considerably hindered. However, the identification of further novel antigens by serological means, could result in the development of new vaccines. The specificity and speed afforded by PCR amplification indicated that this technique showed excellent potential for use in local diagnostic laboratories.
Methods for serotype classification of Haemophilus paragallinarum field isolates.
(1998) Taylor, Kerry Lyn.; Coetzer, Theresa Helen Taillefer.; Horner, Roger F.
Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished.