Listeriosis has the highest fatality and hospitalization rate among foodborne illnesses. Listeria monocytogenes causes listeriosis and is a difficult bacterium for ready-to-eat food processors to eliminate because of its ability to grow in the absence of oxygen and under refrigeration. Recently, milk and its proteins have gained recognition as the largest source of biologically active peptides, and, it stands reason that several antimicrobial peptides (AMP) can be released from casein as it is the most abundant milk protein. AMPs are commonly obtained by cutting the whole protein into peptide fragments using enzymes or by acidification. The objective of this study was to predict potential AMPs through computer aided tools, improve hydrolysate preparation, and determine trypsin and pepsin-casein hydrolysate antimicrobial activity in growth media and on frankfurters against two strains of Listeria monocytogenes (Scott A and 310) and Escherichia coli O157:H7 (Salami strain). The prediction study procedure was to identify the most common variants of primary peptide sequences. The sequences were analyzed for greatest possible enzyme cuts on the protein, peptide masses, isoelectric point, net charge and percent hydrophilic residues using online proteomics programs. The fragments were explored for AMP commonalities: fragment length of 3 to 50 amino acids, positive (cationic) net charge, and hydrophilic residues between 25 and 50%. This technique identified 16 potential AMPs which proved that it is possible to screen for AMPs. The method used to determine the trypsin-casein hydrolysate (TCH) and pepsin-casein hydrolysate (PCH) antimicrobial activity was to hydrolyze sodium caseinate with pepsin or trypsin. L. monocytogenes (strains Scott A and 310) were incubated in 0, 10, 20, and 40% PCH and 0 and 50% TCH concentrations over a 24 hour period. PCH suppressed growth of L. monocytogenes Scott A by 1.76 log CFU/mL and reduced initial populations of L. monocytogenes 310 and E. coli O157:H7 by 0.52 and 0.62 log CFU/ml, respectively. TCH had little or no effect on growth suppression of any of the three test organisms. The frankfurter study was conducted by spot inoculating frankfurters with L. monocytogenes Scott A and then dipping frankfurters into one of five treatments (deionized water, pH 2.7 buffer, pH 5.1 buffer, pH 2.7 PCH, and pH 5.1 PCH) for 30 seconds; inoculated frankfurters that were not dipped served as controls. Frankfurters were incubated at 32°C for seven days. The results showed that there was no significant difference (p>0.05) in antimicrobial effectiveness among the treatments and control. This study demonstrated that enzymatically derived casein hydrolysates somewhat inhibit growth of L. monocytogenes and E. coli O157:H7 in culture media, but were ineffective when applied to frankfurters. Casein hydrolysate solutions can be easily made in a processing facility for application in fluid systems such as an antimicrobial spray on beef carcasses and in milk, juice, sports drinks, soda, soups, and yogurt. It also could be used in solid systems such as frankfurters, cheese, ground beef, and processed or RTE foods.

The Best PhD Thesis in the Faculties of Architecture, Arts, Business &Economics, Education, Law and Social Sciences (University of HongKong), Li Ka Shing Prize, 2008-2009 / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

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.

A variety of proteases have been shown to promote the progression of cancer by virtue of their ability to degrade extracellular proteinaceous barriers, such as basement membrane and interstitial stroma. At the outset of this study available evidence strongly implicated cathepsin D in breast cancer metastasis. It was envisaged that an antibody inhibitory to the activity of this enzyme might retard invasion, and restrain a tumour from spreading. To this end anti-peptide antibodies were generated against a peptide sequence derived from the substrate capturing "flap" of the enzyme. Inhibition of enzyme activity by these antibodies could not be demonstrated, probably due to the lack of a suitably sensitive enzyme assay. However, the rationale of this study and the expertise gained from it could be applied, in the future, to enzymes that have since been found to be more relevant to tumour invasion. A feature of many transformed cells is an anomalous lysosomal enzyme trafficking system, and concomitant hyper-secretion of some enzymes. The distribution of low pH compartments and lysosomal enzyme-containing compartments was investigated in human breast epithelial cells, and their c-Ha-ras- transformed counterparts. Immunofluorescence and immunoelectron microscopy showed that these compartments have a more peripheral cellular distribution with respect to normal cells, and cathepsins B and D were cell surface-associated. Studies were undertaken to reveal the extracellular matrix degrading ability of c-Ha- ras-transformed cells. Transformed cells exhibited increased degradation of fluorescein-labelled extracellular matrix in serum free medium, and increased motility, and degradation and disruption of extracellular matrix in serum-containing medium. In vitro invasion through artificial basement membrane by transformed cells was investigated using scanning electron microscopy, and was further used to preliminarily identify the proteases involved in invasion by specific inhibition. By this means, greatest inhibition of in vitro invasion was obtained using a specific metalloproteinase inhibitor. Overexpression by transformed cells of a metalloproteinase was detected by gelatin zymography. Together these results suggest that the increased invasive capacity of ras-transformed breast epithelial cells may be largely due to increased metalloproteinase activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg , 1996.

Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease affecting young chickens, which is responsible for significant losses in the poultry industry world-wide. The virus specifically infects and destroys B-cell precursors within the bursa of Fabricius, an avian lymphoid organ, leading to immunosuppression. IBDV has a bi-segmented, double-stranded RNA genome. The larger segment encodes a 110-kDa precursor polyprotein, designated NH₂-VPX-VP4-VP3-COOH, in a single open reading frame. The autocatalytic processing of this precursor into mature proteins is a critical step in viral replication and VP4 is the putative protease responsible for this cleavage. This study concerns the development of a strategy to clone and express recombinant VP4 and describes the use of VP4 as a marker for rapid and effective detection of IBDV. VP4 cDNA was produced and amplified by optimisation of a reverse transcription coupled to the polymerase chain reaction (RT-PCR), providing a clear and sensitive assay. Anti-peptide antibodies were raised against a selected peptide from VP4 and were used to probe homogenates of infected bursae for the native protein to assess their potential for immunological detection. These antibody-related results are promising though inconclusive, due to the complex nature of the assayed sample. Amplified VP4 cDNA from KwaZulu-Natal strains of IBDV isolated from 1989 to 1997 was also examined by restriction fragment length polymorphism (RFLP) analysis to determine the relatedness of local IBDV to global strains. All KwaZulu-Natal samples produced identical patterns, which were most similar to one of ten international strains examined, namely, the British strain UK661. Samples infected with IBDV were also probed for VP4 activity. Double basic amino acid cleavage sites have been proposed for the putative protease and infected samples were assayed for activity against the fluorogenic peptide Cbz-Arg-Arg-AMC. Demonstrably higher activity was found in infected versus uninfected samples, although the origin of this activity is unclear. The findings in this study suggest that VP4 warrants further attention, both as a marker for infectious bursal disease, and as a novel viral protease. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Zehn Jahre nach dem Ausbruch des Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV, ist ein neues Betacoronavirus, das Middle East Respiratory Syndrome Coronavirus, MERS-CoV, auf der arabischen Halbinsel entdeckt worden. Seine anhaltende Ausbreitung stellt eine Bedrohung für die öffentliche Gesundheit dar. Das Spike (S) Protein der Coronaviren vermittelt den viralen Eintritt in Wirtszellen und bestimmt wesentlich den viralen Tropismus und die virale Pathogenese. Das Verständnis der Determinanten des MERS-CoV Spike (MERS-S)-vermittelnden Eintritts in Zellen könnte daher wichtige Einblicke in die MERS-CoV-Biologie liefern und war somit das erste Ziel dieser Arbeit. Um den Eintritt in die Zelle zu ermöglichen, muss das Coronavirus S-Protein durch Wirtszellproteasen aktiviert werden, welche potentielle Ziele für die therapeutischen Intervention darstellen. Daher sollten im zweiten Ziel dieser Arbeit Proteasen identifiziert werden, die MERS-S aktivieren. Das S-Protein ist das Hauptangriffsziel neutralisierender Antikörper und experimentelle Systeme zur S-Analyse können für die Diagnostik eingesetzt werden. Das letzte Ziel dieser Arbeit war es daher, die MERS-CoV Seroprävalenz in Saudi Arabien zu ermitteln. Es wurde ein lentivirales Vektorensystem etabliert, welches die Analyse des MERS-S-getriebenen Zelleintritts ermöglicht. Mit Hilfe dieses Systems konnte gezeigt werden, dass MERS-S den Eintritt in ein breites Spektrum humaner Zelllinien, wie Lungen-, Nieren- und Darmzellen vermittelt, was mit der klinischen Manifestation von MERS einhergeht. Der Wirtszelleintritt war unabhängig von bereits beschriebenen Coronavirus Eintrittsrezeptoren, wurde jedoch durch die endosomale Cysteinprotease Cathepsin L und die Transmembranserinprotease TMPRSS2 gefördert. Im Gegensatz dazu war die Aktivität von Proprotein Konvertasen für den S-Protein-vermittelnden Eintritt entbehrlich. Schließlich zeigten Neutralisationstests, dass Seren von Patienten aus der östlichen Provinz Saudi Arabiens, die zwischen 2010-2011 und 2012 entnommen wurden, keine MERS-S-neutralisierenden Antikörper enthielten. Dies deutet darauf hin, dass MERS-CoV-Infektionen vor dem Ausbruch 2012 nur selten vorkamen. Die gewonnen Ergebnisse tragen wesentlich zum Verständnis des MERS-CoV-Eintritts in Zellen bei und liefern wichtige Informationen zur MERS-CoV-Epidemiologie. Weiterhin könnte die Beobachtung, dass der Protease-Inhibitor Camostat, der für den Einsatz im Menschen zugelassen ist (in Japan), TMPRSS2 blockiert und damit den MERS-CoV Eintritt inhibiert, helfen, Behandlungsstrategien für MERS-Patienten zu etablieren.

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014