The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb was purified from bloodstream forms of T. b. brucei by a combination of three phase partitioning (between ammonium sulfate and tertiary butanol), and chromatography on quaternary amine or pepstatin A-Sepharose resins. Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has cathepsin L-like specificity for synthetic substrates and readily degrades various proteins. In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no trypanopain-like activity was detectable in the blood of infected hosts, so it appears that trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host proteins. Antibodies against a peptide corresponding to a region of the trypanopain active site were produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting antibodies were produced and these effects varied with the substrate tested. Thus, the in vivo effects of anti-trypanopain antibodies will only become clearly understood once the physiological substrates of trypanopain have been identified. Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely intracellular target of these inhibitors, indicating that the enzyme is essential for parasite viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus, trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

The collagenolytic proteinases, type IV collagenase and cathepsins Land H, have been implicated in tumour invasion and metastasis, by virtue of their degradative action on the extracellular matrix barriers traversed by migrating tumour cells. Type IV collagenase was isolated from human leucocytes using anti-peptide antibody immunoaffinity chromatography. The highly specific targeting of both native and denatured forms of human type IV collagenase by these anti-peptide antibodies holds much promise for immunolocalisation studies in human tumour tissue. Cathepsin L was purified in both a free; single-chain form from sheep liver, and as complexes with the endogenous cysteine proteinase inhibitor, stefin B. These complexes comprised mixtures of the usual tight-binding non-covalent, inhibitory complexes, and novel, proteolytically active, covalent cathepsin L/stefin B complexes. The latter form spontaneously in a pH-dependent manner in vitro from purified, active constituents. The primary structures of these complexing moieties from sheep liver are reported here for the first time, and showed a high degree of sequence homology with their human counterparts. Single-chain cathepsin L, both in the free, and novel, covalently complexed forms, manifested stability and increased activity at neutral pH, thus suggesting a role in extracellular tissue destruction. This potential involvement in tumour invasion was strengthened by demonstrating that the single-chain form of the enzyme, and similar covalent complexes, active under physiological conditions, could be isolated from liver tissue homogenates of higher primates, baboon (Papio ursinus) and man. A battery of versatile polyclonal anti-sheep cathepsin L and anti-human cathepsins L and H peptide antibodies were raised in chickens and rabbits. The chicken egg yolk antibodies were often of a higher titre than the corresponding rabbit serum antibodies, and additionally manifested unique immunoinhibitory properties. In the case of the polyclonal chicken anti-sheep cathepsin L antibodies, this was derived from their ability to target a peptide located in the active site of cathepsin L. The chicken anti-human cathepsins L and H peptide antibodies constitute the immunological probes of choice for immunolocalisation and in vitro tumour invasion studies to elucidate the relative contributions of these collagenolytic cathepsins to tumour invasion, and could ultimately find application in tumour immunotherapy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1992.

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. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases. Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The conclusion of this study is that, given the high overall conservation of congopain genes in the genome, for the purpose of anti-disease vaccine, it is likely that a single immunogen will suffice to raise antibody able to inhibit all circulating congopain-like cysteine proteases. For chemotherapy however, a more in-depth enzymatic characterisation of the mutants, involving functional recombinant expression, will have to be undertaken. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

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

Fusarium graminearum causes Fusarium Head Blight, (one of) the most destructive cereal diseases in Canada. Yield loss, quality degradation and mycotoxin production make Fusarium a multifaceted threat. Regulated production of reactive oxygen species by Nox enzymes is indispensable for fungal pathogenesis. F. graminearum Nox mutant ∆noxAB produced equivalent mycotoxin but caused reduced virulence than wild-type. We hypothesized that Nox mediated redox signaling may participate in F. graminearum pathogenicity. Two-DE and gel-free biotin affinity chromatography, followed by LC-MS/MS analysis were employed for a comparative redox-proteomics analysis between wild-type and ∆noxAB to identify proteins oxidized by Nox activity. Total 35 proteins, 10 by 2-DE and 29 by gel-free system, were identified. 34% proteins participated in fungal metabolism, 20% in electron transfer reactions and 9% were anti-oxidant proteins. The findings suggested that Nox mediated thiol-disulfide exchange in proteins provide a switch for redox-dependent regulation of metabolic and developmental processes during induction of FHB.

Fusarium graminearum causes Fusarium Head Blight, (one of) the most destructive cereal diseases in Canada. Yield loss, quality degradation and mycotoxin production make Fusarium a multifaceted threat. Regulated production of reactive oxygen species by Nox enzymes is indispensable for fungal pathogenesis. F. graminearum Nox mutant ∆noxAB produced equivalent mycotoxin but caused reduced virulence than wild-type. We hypothesized that Nox mediated redox signaling may participate in F. graminearum pathogenicity. Two-DE and gel-free biotin affinity chromatography, followed by LC-MS/MS analysis were employed for a comparative redox-proteomics analysis between wild-type and ∆noxAB to identify proteins oxidized by Nox activity. Total 35 proteins, 10 by 2-DE and 29 by gel-free system, were identified. 34% proteins participated in fungal metabolism, 20% in electron transfer reactions and 9% were anti-oxidant proteins. The findings suggested that Nox mediated thiol-disulfide exchange in proteins provide a switch for redox-dependent regulation of metabolic and developmental processes during induction of FHB.

Self-assembled monolayers (SAMs) derived from long chain alkanethiols are known to exhibit generalized trends as a function of chain length where n denotes the number of methylene units (CH2). For n 3, these trends are no longer manifest. It can be shown that SAMs of short chain lengths are much more affected by the presence and type of functional group. The reduction of electrochemically induced SAMs derived from cysteine (cys), cystine ((cys)2), mercaptopropionic acid (MPA) and mercaptoethylamine (MEA) from Au(111) highlight the effect of the two functional groups evaluated (R-CO2 - and R-NH2). The reductive desorption of these species was monitored by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) in 0.1 M KClO4 and 0.1 M NaOH. The work presented herein demonstrates that under short time frames of immobilization, the presence of NH2 provides a stabilizing effect to the SAM. Cys and (cys)2 SAMs that maintain both functional groups are generally found to provide the lowest surface coverage under the short term conditions of assembly. The thiol derived monolayers (cys) are consistently higher packed than the disulfide SAMs iv from (cys)2 in both media evaluated. In 0.1 M NaOH however, cys coverage is consistent with coverages obtained from very long incubation times. In the presence of the strong base the disulfide species, (cys)2, desorbs at potentials that are always more positive than those of the thiol species (cys), further supporting poor monolayer formation. Additionally, these monolayers also exhibit the presence of two separate processes in 0.1 M KClO4, whereas only desorption is noted in 0.1 M NaOH. It is likely that a deprotonation of the amine group occurs prior to the desorption of the SAM. The SAM desorption occurs near -0.65 V vs. SCE, and the de-protonation at about -0.50 V vs. SCE. Since the monolayers formed from cys are better formed than those from (cys)2, this deprotonation is much more pronounced in the cys SAMs. The presence of only the CO2 - group (MPA) on the SAM, yields surface coverage that is intermediate compared to the bi-functionalized SAMs formed from cys and (cys)2 and the NH2 containing SAMs of MEA. In the potential region up to and prior to desorption, only one process is noted in both media. SAMs derived from MEA provide the highest surface coverage of the four species, approximating theoretical values. The presence of two surface species is observed in both media, as a result of trans and gauche binding. Of the four species evaluated, MEA appears to be most suitable for rapid SAM formation. The disulfide species, (cys)2, is found to be unsuitable for short-term preparation of SAMs.

[Truncated abstract] The placenta forms a barrier regulating the transfer of gases, nutrients and wastes between the mother and the developing conceptus, and also produces hormones affecting both the fetus and the mother. This barrier is formed by the differentiation of the outer layer of the blastocyst- the trophoblast- to facilitate implantation and subsequent invasion of the uterus. The trophoblast consists of an underlying proliferative pool of cytotrophoblasts, which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast that forms the barrier between the mother and fetus. Moreover, the location of the syncytiotrophoblast directly in contact with the maternal circulation suggests an endothelial role for the trophoblast regulating blood flow, thrombosis and immune cell adhesion. Disruption to the function of the human trophoblast may result in preeclampsia, a maternally manifested disorder of pregnancy characterised by hypertension and proteinurea. Blood flow to preeclamptic placentae is reduced and the cytotrophoblast pool is diminished; however the exact cause (or causes) remains elusive. Many potential causes are hypothesised, including endothelial damage, premature remodelling of maternal spiral arteries, increased oxidative stress and impaired trophoblast differentiation and apoptosis. Caspase-14 is an unusual caspase in that it is not involved in apoptosis. Furthermore, it possesses a limited, predominantly epithelial, tissue distribution. In the epidermis, caspase-14 is expressed in the apical differentiating layers. Here it cleaves profilaggrin to stabilise intracellular keratin intermediate filaments, and indirectly provides natural hydration and UV protection to the corneocytes. Thus, caspase-14 is vital to the maintenance of the barrier function of the skin. ... As differentiation-associated genes were elevated in the absence of caspase-14, this implies that caspase-14 suppresses biochemical trophoblast differentiation. The cytoskeletal keratin network was also examined following RNA Interference. The synthesis of cytokeratin 18 was significantly enhanced after caspase-14 suppression during BeWo differentiation, linking caspase-14 with keratin homeostasis. Therefore caspase-14 suppresses trophoblast differentiation, potentially through modulation of the cytoskeletal keratin filament network. The precise mechanism remains to be elucidated, however the identification of pathways regulated by caspase-14 advances our knowledge of trophoblast differentiation and potential causes of disorders of pregnancy. In summary, caspase-14 appears to be involved in the suppression of differentiation in the human trophoblast. As disorders of pregnancy such as preeclampsia often feature disturbed differentiation and a diminished cytotrophoblast pool, a greater understanding of caspase-14 biology in the human placenta could lead potential therapies for various disorders of pregnancy.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.