LITERATURE REVIEW
African trypanosomosis
Areas where no cases were reported are shown in green (Franco et al., 2017). B) Distribution of important animal infectious trypanosomal species in Africa. Multiple species and subspecies of animal infectious trypanosomes circulate in cattle within HAT zones (Fyfe et al., 2017) (Fig. 1.1, panel B).
Trypanosome classification
3 losses are experienced due to mortality and reduction in calving rate, draft and meat and milk production due to AAT infections (Swallow, 2000).
Parasite structure and genomic organisation
VSG and antigenic variation
A single VSG is expressed as only a single VSG allele at any given time and can be transcribed from the VSG repertoire (Pays et al., 2006). Each VSG ES has polycistronic transcription units that contain expression site-associated genes (ESAGs) along with the VSG gene (Pays et al., 2001).
Life cycle of the trypanosomal parasite
Short epimastigotes attach to epithelial cells in the salivary gland and can reproduce by asymmetric division to produce metacyclic trypomastigotes (Matthews, 2005). Metacyclic trypomastigotes regain the VSG envelope and are released into the lumen of the salivary gland, where they can be transmitted to the mammalian host; this completes the life cycle (Rotureau et al., 2012).
Immune response
The hypothetical interaction between IgM (turquoise) and the N-terminus of the immunogenic VSG (dark blue) molecules (Mugnier et al., 2016). 9 the cytokine network by using the cytokines as growth factors and modifying the effector functions of the immune system (Hide et al., 1989).
Control, diagnosis and chemotherapy for trypanosomosis
- Control
- Diagnosis
- Chemotherapies
The presence of parasites in the CSF is indicative of second stage infection (Chappuis et al., 2005). Currently used chemotherapies for AAT are diminazene aceturate (Berenil®), isometamidium chloride (Samorin®) and homidium chloride (Novidium®) (Geerts et al., 2001).
Peptidase virulence factors
- Metacaspases
- Structure of metacaspases
- Functions of metacaspases
- Oligopeptidase B
- Functions of oligopeptidase B
The C-terminal domain of LmjMCA interacts with proteins involved in stress regulation (Casanova et al., 2015). Taken together, this highlights the potential of MCAs to act in important regulatory processes (Coll et al., 2010).
Structure-based drug design
Antibody production using phage display
- Antibody formats produced by phage display
- Applications of phage display
In 2004, one such phage display library, using chicken immunoglobulin genes, was developed and named the Nkuku® library (van Wyngaardt et al., 2004). The Nkuku® phage display library was used to map the epitopes of the SAT2 foot-and-mouth disease virus in an attempt to neutralize the virus (Opperman et al., 2012).
Objectives of current study
Antibodies are used directly in diagnostics for the detection of pathogen antigen (Dussart et al., 2008) and for competition with serum antibodies for binding to the pathogen antigen (Dong et al., 2007). Single chain variable fragment antibodies can be used in many standard immunodiagnostic tests (Thompson and Neiman, 1987; Nissim et al., 1994; . van Wyngaardt and Du Plessis, 1998).
Introduction
This extended domain is present in each of the single-copy MCAs in both Trypanosoma spp. The multicopy MCAs with mutations of the catalytic Cys (purple) and both catalytic His and Cys (red) were blocked in their respective colors.
Materials and methods
The plasmid DNA from the recombinant colonies was isolated using the GeneJet™ Plasmid Miniprep Kit, according to the manufacturer's instructions. Subcloning of the TbbMCA2 and TviMCA5 gene constructs into the bacterial pET-28a and pET-32a expression plasmids.
Results
Subcloning of the TbbMCA2 and TviMCA5 genes into the bacterial pET-28a and pET-32a expression plasmids. Removal of the C-terminal domain (21.5 kDa band) from the 77 kDa full-length protein will produce the 38 kDa protein. Subsequent cleavage of the N-terminal domain of the 38 kDa protein would result in the 29 kDa protein.
Cleavage of the C-terminal domain of the full-length protein may result from the 37.5 kDa protein eluted in fractions 3 and 4.
Discussion
The cleavage of the N-terminal domain of the 66.4 kDa protein can result in the 43.9 kDa protein consisting of the catalytic domain and the C-terminal domain. It was shown that the full-length protein was expressed in the insoluble fraction, but was also expressed in the soluble fraction after removal of the N-terminal 6xHis tag. This cleavage pattern, which resulted from automatic processing, was thought to result from the separation of the different domains to release the catalytic domain.
It was suggested by Moss et al. 2007) that the cleaved domains were still part of the mature protein structure and was confirmed by McLuskey et al. 2012) where they were shown to still be covalently linked to the main body of the protein.
ENZYMATIC CHARACTERISATION OF NATIVE AND
Introduction
Due to their presence in all kingdoms except the metazoan kingdom, and their potential involvement in similar roles to those of the caspases, the MCAs have been identified as virulence factors. Thus, their involvement in cell death, cellular processes and absence in the mammalian hosts make the MCAs attractive targets for the development of new chemotherapeutics. However, to date, there have been no reports of the molecular characterization of any MCA5 from the animal infectious T.
In addition, calcium has been shown to play an important role in the self-processing capacity and/or peptidolytic activity of MCA.
Materials and methods
The optimal enzyme concentration for the hydrolysis of the fluorogenic peptide substrate was determined by varying the concentration of the enzyme (0–5 µg/ml recombinant, 0–10 µg/ml native, in 10 µl) and incubation with MCA assay buffer containing 10 mM Z-Gly-Gly-Arg-AMC (90 µl), in triplicate, and fluorescence measured as described above. During this period, 1, 10 and 100 µM of the panel of reversible and irreversible inhibitors, prepared in MCA assay buffer, were added in triplicate to the wells of a black FluoroNunc™ 96-well plate. The AMC standard curve (Appendix B2) and the rate change were used to determine the resulting inhibition of the enzyme, which was reported as the percent inhibition compared to the uninhibited enzyme.
The AMC standard curve (Appendix B2) and the change in rate were used to determine the resulting inhibition of the enzyme, which was reported as a percentage of inhibition compared to that of the uninhibited enzyme.
Results
From the literature, autoprocessing results in the cleavage of the N- and C-terminal domains to release the catalytic domain from the full-length protein. The eluted fractions of TcoMCA5C202G and TviMCA5 mutants were counted silver-stained. Hydrolysis of the Z-Gly-Gly-Arg-AMC substrate was detected only in the second fraction of the TcoMCA5H147AC202G double mutant purified using nickel affinity chromatography.
The resulting fluorescence from hydrolysis of the AMC fluorophore in 60 min was measured at Ex360nm.
Discussion
First, C-terminal cleavage at Arg298, followed by N-terminal cleavage at Arg63, Arg79, and Arg135, is required for the release of the active catalytic domain ( Zalila et al., 2011 ). Removal of the N-terminal domain would result in the 31 kDa catalytic domain which is close to the expected size of TcoMCA5 and TviMCA5. No significant differences in autoprocessing patterns were observed with expression of WT and catalytic residue mutants.
Deletion of the C-terminal domain of the full-length protein is thought to produce the 45 kDa protein.
DOCKING STUDIES OF MCA2 INHIBITORS WITH MCA2 FROM
Introduction
Various methodologies are followed in the discovery of new chemotherapies (Matthews, 2015; Field et al., 2017). A positive outcome of SBDD methodologies is the identification of the important residues involved in binding, which can inform modification of existing ligands to achieve greater binding affinity for the target (Jain et al., 2011; Blaney, 2012). . The 3D structure of TbbMCA2 has since been solved ( McLuskey et al., 2012 ), but the binding site and affinity of the Berg ligands for the TbbMCA2 target has not been established.
In this study, validation of ligand binding from a previously designed Berg library to the active site of the 3D structure of TbbMCA2 (resolved by X-ray diffraction (McLuskey et al., 2012)) was performed using molecular docking software.
Materials and Methods
This residue in each of the four TbbMCA2 models was changed back to Cys to best simulate the active site environment. Strongly bound water molecules are often conserved in multiple crystallographic structures (Ferreira et al., 2015), as is evident for TbbMCA2, where three conserved water molecules line the bottom of the active site in all four 3D structures (McLuskey et al., 2012). Most docking algorithms work by attaching flexible ligands to the rigid binding site of a protein "receptor".
Using the default settings in the CDocker protocol of Discovery Studio (Dassault Systèmes BIOVIA), the ligands from both the Berg and the commercial libraries were in the binding site of the 3D structures of.
Results
PDB files of the resulting (A) TbbMCA2 4AF8 and homology (B) TcoMCA5 and (C) TviMCA5 structures were individually loaded into ProSA software for evaluation (Wiederstein and Sippl, 2007). The surface area of the active site of TbbMCA2 4AF8 in the four aqueous preparations is depicted in Fig. 105 Table 4.4: -Cdocker interaction energies for the Berg library ligands that were successfully docked into the active site of the four water preparations of TbbMCA2 4AF8.
In general, similar -Docker interaction energies were obtained for the Berg library ligands when docked to the active sites of TbbMCA2 4AF8, TcoMCA5, and TviMCA5 with an “all water” preparation.
Discussion
The application of molecular binding studies has enabled investigations into ligand - 'receptor' interactions as well as elucidation of the mechanism of action (Njogu et al., 2016). Common inhibitor scaffolds were identified along with predicted conformations within the active site and inhibitor interactions with the cruzipain 'receptor' ( Salas-Sarduy et al., 2017 ). Ligand binding to the protein “receptor” requires rearrangement of water molecules surrounding both the ligand and the “receptor” (Lounnas et al., 2013).
Interactions between the ligands and the protein 'receptor' were determined using the results of the docking studies.
PHAGE DISPLAY AND ANTIGEN DETECTION ELISA OF
Introduction
The established method of producing polyclonal antibodies is through animal immunization (Schirrmann et al., 2011). Consequently, there will be no deviations between different groups as experienced with animal serum (Schirrmann et al., 2011). The naïve Nkuku® library is not restricted to any specific target and consequently facilitates the generation of monovalent scFv antibodies against an almost unlimited range of antigens (van Wyngaardt et al., 2004).
Manni kitaabaa Nkuku® jiiniiwwan immunoglobulin V irra deebiin qindaa’an seelii B (IgM) hanqaaquu arjoomtoota hin talaalamne irraa walitti qabame (van Wyngaardt et al., 2004).
Materials and methods
Furthermore, homology modeling of the scFv and its linkage to the OPB antigen is reported. A 1:100 dilution of the overnight culture was performed in fresh 2xYT, without antibiotic, and incubated at 37°C until the OD600 reached 0.5. Digestion samples (5 µl) were electrophoresed on a 3% (w/v) agarose gel containing ethidium bromide (0.5 µg/ml) in 1xTAE buffer.
Single colonies from the selected scFv clones were used to inoculate 2xYT (5 ml), without antibiotic, and incubated at 37 °C at 220 rpm for 16 h.
Results
The alignment of the scFv sequences is shown along with the Qmean scores indicated in blue (high quality prediction) and red/orange (low quality prediction). Detection of the 74 kDa pIII fusion protein using the anti-M13 antibody (phage coat protein) was unsuccessful (not shown). Detection of the scFv::pIII fusion protein using the anti-M13 antibody was unsuccessful (not shown).
Despite this, the absorbance values of the osmotic shock fractions were still higher than those of the controls.
Discussion
Panning of the Nkuku® library was performed to select scFv antibodies specific for TcoOPB and TviOPB. Successful purification of the 70 kDa myc-tagged scFv::pIII fusion demonstrated that the OPB-specific scFv was indeed able to detect OPB. This may mean that the scFv has a lower OPB detection limit compared to polyclonal antibodies.
As such, the anti-myc antibody was used to detect bound scFv when used as a capture antibody.
GENERAL DISCUSSION
In the early years of MCA research, it was thought that the MCAs functioned similarly to the caspases in cell death (Madeo et al., 2002). In the mitochondrial matrix, full-length LmjMCA is extensively processed into fragments containing the catalytic domain (Zalila et al., 2011). The activity of the catalytic domain increased the sensitivity of the parasite to H2O2 by weakening the mitochondrion (Zalila et al., 2011).
This peptidase plays a role in the cell cycle and is also a virulence factor that is secreted into the bloodstream of the mammalian host (Proto et al., 2011).
