Masters Degrees (Biochemistry)

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Recombinant expression, purification and characterisation of TzCATB and TzstefinB from Trichinella zimbabwensis.
(2024) Woodgate, Ryan.; Coetzer, Theresa Helen Taillefer.
Parasitic roundworms of the genus Trichinella, the causative agents of Trichinellosis, are highly successful parasites evident by their broad geographical and host ranges and their ability to manipulate the host immune system. The focal species of this study was T. zimbabwensis; a lesser studied member of the genus prevailing in Sub-Saharan Africa. Although not yet reported in humans, in the absence of a diagnostic test for this species of Trichinella, it poses a risk to human health and economic well-being. Research on different antigens expressed across all the stages of the parasite lifecycle may identify diagnostic, drug and vaccination targets. Cysteine proteases and protease inhibitors are particularly valuable targets for these purposes due to their essential functions in the parasite’s lifecycle, such as penetration of the small intestinal wall, nutrient acquisition and immune evasion amongst others. The aim of the present study was to recombinantly express and characterise cathepsin B (TzCATB) and stefin B (TzstefinB) from T. zimbabwensis. Recombinant TzCATB was expressed in the pET-28a and pCold-1 expression vectors as insoluble proteins at 43 and 42 kDa, respectively. After solubilisation and nickel immobilised metal affinity chromatography (Ni-IMAC) purification of these proteins, no proteolytic activity was detected. Recombinant TzCATB was subsequently expressed in the pCold-TF expression vector as a soluble protein 90 kDa protein, which was purified using immunoaffinity chromatography and the attached trigger factor fusion protein removed with thrombin cleavage. However, this preparation of TzCATB also did not have hydrolytic activity but was detected by chicken anti-TzCATB IgY antibodies in a western blot, but not by rabbit anti-human cathepsin B antibodies. Recombinant TzstefinB was expressed in the pET-28a expression vector as a soluble 16 kDa protein and purified with IMAC. Purified TzstefinB was used to raise IgY antibodies in chickens, which were confirmed to detect the protein in ELISA and western blotting. These antibodies did not detect sheep stefin B. Purified TzstefinB inhibited the hydrolysis of Z-Phe-Arg-AMC by the plant cysteine protease papain as well as by cathepsin L from the parasite Trypanosoma congolense. The inhibition of papain by TzstefinB was also detected with reverse zymography. The inhibitory activity of recombinant TzstefinB was shown to be stable from pH 5.0 to pH 9.0, was rapidly inactivated with incubation at 99 ºC, and was retained after storage at both 4 ºC and room temperature for up to 3 weeks. Thus, TzCATB was recombinantly expressed but was inactive, TzstefinB was recombinantly expressed and demonstrated to have inhibitory activity that is stable across a broad pH range but sensitive to high temperature, and both of these proteins have potential as diagnostic markers.
Investigating the impact of using intravaginal products on cervical lesions, genital inflammation, and curable sexually transmitted infections/ genital infections in adolescent girls and young women in KwaZulu-Natal.
(2024) Radebe, Phumla Londeka.; Gumbi, Pamela Phumelele.; Passmore, Jo-Ann.; Sibeko, Singeziwe.
Background: Vaginally inserted products (VIPs) are used to cleanse, enhance sexual pleasure or modify the female genital tract to a desired state by young South African adolescent girls and young women (AGYW), in regions of the country that practice “dry sex”. This study hypothesized that sexual immaturity in adolescent females in conjunction with VIP use would exacerbate injury to the cervicovaginal mucosa and increase vaginal inflammatory responses, which may influence the risk of acquiring human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs). Methods: Sexually active and HIV-uninfected cisgender adolescent girls (n=188, 14-19-years old) and adult women (n=64, 25-35 years old) were enrolled in an HIV endemic rural Vulindlela area, in KwaZulu-Natal (KZN), South Africa. A detailed questionnaire was used to collect demographic, sexual behaviour, and vaginal practices data. Cervical ectopy and other cervical abnormalities were identified by colposcopy. Luminex assay was used to measure the concentrations of various inflammatory cytokines from cervicovaginal secretions. Human papillomavirus (HPV) genotyping was done using a DNA Flow hybridization system. Participants were also tested for the presence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), bacterial vaginosis (BV), yeast and fungal hyphae. Results: This study found that adult women were more likely to be involved in risky sexual behaviour compared to adolescents, although the prevalence of STIs was higher in adolescents compared to adults, particularly CT (p<0.001). Colposcopic observations showed that adults were more likely to have signs of cervical injury and trauma compared to adolescents (p<0.0001), and significantly higher cervicovaginal concentrations of interleukin (IL-)8 (p<0.0001), granulocyte-colony stimulating factor (G-CSF) (p<0.0001) and IL-10 (p<0.0001) compared to adolescents. However, tumour necrosis factor alpha (TNF-) concentrations were significantly higher in adolescents compared to adults (p<0.0001). The use of vagina stimulating products was common in this cohort, with adolescents reporting a wider range than adults. These products were either traditionally made or available commercially, taken orally, or applied internally or externally in the vagina. According to the adolescents and women, the products were used for different reasons and the preferences by age group were different: Adolescents most commonly used alum (a combination of aluminum sulphate and potassium sulphate) while adults most commonly used “ibhodwe labafazi” (translated from Zulu to mean “ladies pot”; a commercially available scented petroleum jelly with unknown ingredients). Adolescents who used alum were likely to have injury signs compared to adolescents who did not use any products (p=0.002). In addition, adult women who used “ibhodwe labafazi” were 20 times more likely to have cervical ectopy compared to adult nonusers (p=0.004). Among adolescents, the users of “ibhodwe labafazi” had significantly elevated cytokine concentrations, remarkably so for IL-1α (p=0.0223), vascular endothelial growth factor (VEGF) (p=0.0198) and IL-17 (p=0.0150) compared to nonusers. However, in adults, there were no remarkable changes in genital tract cytokine concentrations in users compared to nonusers. However, the use of the products did not appear to have a direct link with the prevalence of infections. Conclusion: In adolescents, the use of alum and “ibhodwe labafazi” may cause injury and inflammation, respectively, which are known to increase the risk of HIV acquisition. In addition, in adult women, using “ibhodwe labafazi” were more likely to have ectopy, which is associated with STIs and HIV risk. These findings need to be confirmed by more extensive cohort studies and suggest that the use of some of the vaginal enhancing products may partly contribute to differences in biological risk profiles that influence HIV susceptibility.
Investigating the role of Lactobacillus isolates from South African young women in the control of bacterial vaginosis (BV) - associated Gardnerella vaginalis and Candida.
(2024) Nomnganga, Busisiwe.; Gumbi, Pamela Phumelele.
Background: Lactobacillus (L.) strains offer a promising approach for preventing and treating viral, bacterial, and fungal infections, serving as alternatives or complements to traditional antibacterial and antifungal therapies. Current treatments for bacterial vaginosis (BV) often entail side effects such as gastrointestinal disturbances, antibiotic resistance, and high recurrence rates, while antifungal treatments may lead to skin irritation and gut health issues, particularly in pregnant women. These challenges underscore the urgent need for alternative strategies to manage vaginal infections. This study investigated the probiotic characteristics of Lactobacillus species isolated from healthy African women and assessed their effects on pathogenic microbes, specifically Gardnerella (G.) vaginalis and Candida (C.) albicans, alongside their production of antimicrobial substances, including lactic acid and H₂O₂. Methods: Thirteen strains of vaginal Lactobacillus species (L. crispatus, L. gasseri, L. jensenii, L. mucosae, and L. vaginalis) were pre-isolated from HIV-negative young women from South Africa. The cell morphology of the bacterial strains was confirmed with the Gram staining technique. The ability of Lactobacillus strains to acidify culture media was measured by monitoring the pH changes over time during incubation using a calibrated pH meter. The production of antimicrobial compounds, specifically D- and L-lactate and hydrogen peroxide (H₂O₂), were quantified using colorimetric assay kits. The inhibitory activity of the isolated Lactobacillus strains against G. vaginalis and C. albicans was assessed using standardised in vitro assays, which typically involved co-culturing the Lactobacillus culture supernatants (LCS) with the pathogens and measuring growth inhibition by optical density (OD600nm) over time. The study compared differences in inhibitory effects, lactic acid, H₂O₂ production, and pH reduction among the Lactobacillus species and strains. The Mann-Whitney non-parametric t-test was used to analyse the data for two groups, while the Kruskal-Wallis one-way ANOVA unpaired test was used with the unadjusted Dunn’s multiple comparisons tests. Results: The growth kinetics of the Lactobacillus isolates were comparable, with their optimum growth reached at 48 to 72 hours. The pH of MRS cultures decreased over time from 6 to 3.5 by all the vaginal isolates. The isolates produced relatively low but measurable H₂O₂, D- and L-lactic acid concentrations ranging from 253 to 440 μMol/L, 1.05 to 2.9 ng/μL, and 0.09 to 0.13 mM, respectively. Lactobacillus crispatus (70.7pa and 94.79pa) showed better production of metabolites, followed by L. gasseri and L. jensenii (95.1pa). Lactobacillus gasseri and L. crispatus demonstrated vigorous antibacterial activity against G. vaginalis, decreasing its growth to about 50%. Candida albicans grew to approximately 70% in the v presence of L. crispatus (70.7pa and 94.79pa) and L. vaginalis 88.5b, showing better inhibitory activity than the other tested Lactobacillus strains. Conclusion: This study highlights the significant potential of African-derived vaginal Lactobacillus species as probiotics for preventing and treating vaginal infections, with L. gasseri and L. crispatus showing the most potent properties. These strains effectively inhibited pathogenic microbes and produced higher amounts of lactic acid and H₂O₂, essential for maintaining a healthy vaginal environment. The results indicate that probiotic efficacy is both species- and strain-dependent, underscoring the need for alternative treatments given the limitations of current therapies.
The isolation and characterisation of proteases from Euphorbia tirucalli, E. triangularis, and Carica papaya latex.
(2023) Boodhoo, Akira.; Coetzer, Theresa Helen Taillefer.
Plant proteases play an important role in the food and industrial sectors from meat tenderisers to milk clotting agents and even anti-parasitic agents. Proteases have been identified in plant latex, but many proteases have not been isolated and characterised. This research aimed to isolate and characterise proteases from the latex of Euphorbia tirucalli, E. triangularis, and Carica papaya. Three-phase partitioning (TPP) of E. tirucalli plant latex revealed the presence of two active proteases on gelatin-containing zymograms, that were subsequently separated by size exclusion chromatography. These proteases were classified as a 75 kDa serine protease (E. tiru SP), inhibited by PSMF, and SBTI and a 37 kDa cysteine protease (E. tiruCP), inhibited by E-64. Analysis of E. triangularis latex by TPP and p-aminobenzamidine affinity chromatography showed the presence of three serine proteases inhibited by PMSF and SBTI, E. laris SP1 (>97.4 kDa), E. laris SP2 (68 kDa) and E. laris SP3 (38 kDa). These proteases showed stability in constant ionic strength buffers from pH 4 to 9, both with and without the presence of the reducing agent cysteine. Papain was isolated from the latex of Carica papaya for use as a control protease in zymograms on account of the anomalous behaviour of commercial papain preparations on these gels. Papain was isolated by two methods: ammonium sulfate precipitation followed by TPP and CM-cellulose cation exchange chromatography. The isolated papain was detected by rabbit anti-papain antibodies in a dot blot and western blot and showed inhibition by E-64. The latex from all three plant species showed milk clotting activity with higher activity in the presence of calcium chloride. These findings suggest that isolated plant latex proteases from the Euphorbia species can be used in the food industry as milk clotting agents. Further characterisation of these isolated proteases should identify further uses in biotechnology.
Structural characterization of caseinolytic protease (ClpP) from Klebsiella pneumonia.
(2023) Zuma, Sbahle Naledi.; Khoza, Thandeka Ntokozo.
Abstract available in PDF.
Isolation and characterisation of umbilical cord blood serum exosomes.
(2023) Mkhize, Sphesihle.; Niesler, Carola Ulrike.
Abstract available in PDF.
Antioxidative and antidiabetic activity and phytochemicals, analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.
(2021) Zuma, Lindiwe Khumbuzile.; Pooe, Ofentse Jacob.
Microbial enzymes have been described as an underutilized source of novel enzymes with potential economic advantages. Recently discovered enzymes such as DNA ligase from metagenomic studies, have been shown to achieve great potential in transforming the reagent market specifically in the African continent. Reagent proteins are frequently utilized in the research field widely and are prone to protein degradation and shelf-life reduction. Hence, this study sought to improve biological activity, shelf life and stability of the two DNA ligases identified from Kogelberg Biosphere metagenomics library. Two recombinant DNA ligases expression studies were done using E.coli BL21 and purification studies were done subsequently using affinity chromatography. Both recombinant DNA ligases (Ligsv081 & LigpET30) were successfully expressed and purified as homogenous proteins. In this study two approaches were used to enhance the biological DNA ligases, the first approach used was PEGylation. The purified proteins were conjugated to PEG using site-specific PEGylation and non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze the secondary structure of the PEG conjugated DNA ligases. Thermal stability assays were then employed to assess protein stability in the conjugation with PEG. Site-specific PEGylation enhanced ligase activity and reduced the formation of protein aggregates. The second approach involved DNA ligase co-expression in the presence of PfHsp70 or chimeric transcription factor, respectively. Protein co-expression and co-purification assays were conducted. The co-expression and co-purification assays of both proteins with chimeric transcription factor (cTF) were successful, followed by co-expression and co-purification of LigpET30-PfHsp70. Ligation assays were conducted to assess bioactivity of proteins. All DNA ligase complexes were functional and their melting point was increased. Taken together, site-specific PEGylation and protein co-expression with PfHsp70 potentially extended the shelf-life and stability of the proteins. PEGylation strategies and co-expression strategies can potentially be used to enhance reagents in diagnostic and therapeutic tools in molecular biology field.
Antioxidative and antidiabetic activity and phytochemicals analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Idris Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
The use of zebrafish to assess water quality and remediation efforts.
(2023) Zondi, Thandolwethu Beauty.; Hewer, Raymond.
Although wastewater effluents continue to be significant polluters of aquatic ecosystems in developing countries with limited water resources, little is known about the ecotoxicity induced by these effluents on fish throughout their early life stages. Several wastewater treatment plants (WWTPs) in South Africa (SA) do not adequately meet the minimal wastewater treatment requirements established by the country's Department of Water and Sanitation (DWS). Moreover, contaminants of emerging concern (CECs) originating from synthetic or natural sources, are widely distributed in aquatic environments of SA. This includes a broad range of natural and chemical compounds, such as aspirin (44243 ng/L), Fluoroquinolones (27100 ng/L), Atenolol (25900 ng/L), Nalidixic acid (25234 ng/L) and Ciprofloxacin (20514 ng/L). In addition to chemical compounds, endocrine disrupting chemicals, pharmaceuticals and personal care products are also distributed in the water systems. In the process of wastewater treatment, agents such as flocculants, coagulants, chemical precipitants (e.g., calcium hydroxide or sodium hydroxide) and chlorine disinfectants are utilized in wastewater treatment settings. However, research to understand the adverse effects that can be caused by these agents on aquatic organisms is still ongoing in SA. In order to bridge this knowledge gap, advanced techniques could be employed to help reveal adverse effects of wastewater as well as any shortcomings of current water remediation techniques. Using an appropriate aquatic model organism with highly conserved physiological pathways present in higher vertebrates (including humans), a rich behavioural repertoire, and occurrence in a variety of habitats would be a novel approach. To this effect, this study employed zebrafish with the aim to monitor six distinct wastewater samples from various regions of SA and to assess the effectiveness of currently used water remediation techniques such as chlorination. Two wastewater effluents, namely, Southern Works Final Effluents (SWFE) and Jacob’s Incoming (JB) alerted potential toxicity during chemical characterization with suboptimal pH (SWFE = 9.02 ± 0.16 and JB = 5.65 ± 0.02) and total alkalinity of zero (0 mg/L) detected for both effluents. The lethal toxicity of these effluents was seen by the elevation of mortality rate up to 77 ± 2.89 % and 100 ± 0.00 %, respectively for SWFE and JB at 40 %, with corresponding LC50 values of 17.77 % and 16.46 %. The zebrafish jaw and face, heart, brain, fins, notochord, somite and tail were significantly deformed (p < 0.05) post-exposure to these effluents, as revealed by morphological scores upon the analysis of the zebrafish’s body structure. Moreover, there was a delay in development due to the aforementioned effluents, unsuccessful hatching, craniofacial abnormalities, pericardial and yolk sac oedema, notochord abnormality somite defects and spinal cord curvature. In addition, locomotor activity of zebrafish was inhibited following observation of distance travelled, frozen moments, acceleration rates, swimming trajectories and exploration rate. Surprisingly, safety of these wastewaters was restored by chemical precipitation revealing non-lethal pH ranges of 6.02 - 8.02 and 6.65 - 7.65 for SWFE and JB, reducing the mortality rate to non-significant levels (p > 0.05) compared to the control. Also, sodium bicarbonate (NaHCO3) at 120 mg/L was found effective at supplementing the wastewater total alkalinity. In contrast, Amanzimtoti water before and after chlorination (TB and TA), Incoming Badulla (IB) and Chatsworth Incoming (CI) exhibited no consistent lethality effects on zebrafish and induced no apparent stress as demonstrated by insignificant expression (p > 0.05) of the stress protein: heat shock protein 70 (HSP70). However, the insignificant mortality v rate (p > 0.05) in the water tested before (TB) and after (TA) chlorination appeared to be the same (~25 %) indicating that chlorination is not enough at completely remediating wastewater. Our study is a pioneer in evaluating the ecotoxicological impact of wastewater effluents from localized regions of a developing country like South Africa in relation to the adjustment of water quality parameters for the neutralization of contaminants. To better understand emerging contaminants released as effluents in SA's water bodies and their interactions with aquatic organisms at the adult stage, more studies needs to be developed.
Quantitative detection and inactivation of Mycoplasma hyopneumoniae.
(2023) Wei, Yanna.; Khoza, Thandeka Ntokozo.; Xiong, Qiyan.
Mycoplasmal pneumonia of swine (MPS) caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) is a chronic respiratory infectious disease of swine and has a significant impact on the economy. The main clinical symptom of the disease is cough, accompanied by reduced growth performance. Inactivated vaccine is used extensively to control M. hyopneumoniae infection worldwide. This study focused on a quantitative detection method and inactivation kinetics of M. hyopneumoniae in the process of vaccine production. In this study, an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was established and optimized using elongation factor thermo unstable (EF-Tu) as the target protein. EF-Tu protein is abundant on the surface of M. hyopneumoniae. The ic-ELISA assay was used to quantify the M. hyopneumoniae cultures at different growth stages, and compared with the traditional color changing unit (CCU) assay. The ic-ELISA assay results obtained for a growth curve were similar to that of the CCU assay indicating a strong correlation in the log phase. Also, a linear regression equation was established between the ic-ELISA and CCU assays in the log phase. The ic- ELISA method was used to evaluate the relative potency values of different batches of cultures over the internal reference vaccine to determine whether the cultures meet the antigen amount requirements for vaccine preparation. Compared with the CCU assay, the established ic-ELISA assay can quantitatively detect the antigen content in the process of vaccine production more quickly. Furthermore, this study also systematically compared the inactivation effects of, formaldehyde, thimerosal, β-propiolactone (BPL) and binary ethylenimine (BEI) on M. hyopneumoniae. Complete inactivation were achieved by 0.01% formaldehyde for 24 h at 37°C, 0.0008% thimerosal for 12 h at 37°C, or 0.02% BPL for 24 h at 4°C or 0.004% BEI for 24 h at 37°C. The immunogenicity of the antigens after inactivation was detected by mice immune test. The IgG antibody level of the mice immunized with the vaccine inactivated by 0.01% formaldehyde for 24 hours was significantly higher than those of the mice immunized with the vaccines inactivated by other solutions. The data indicated that formaldehyde can be used to inactivate M. VIII hyopneumoniae before vaccine preparation. In a conclusion, this study provides important reference value for the antigen quantitative detection and inactivation of M. hyopneumoniae for vaccine production.
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.
Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.
(2022) Ramjeawon, Bhavana Roshenlal.; Coetzer, Theresa Helen Taillefer.
Humans and animals in sub-Saharan Africa are at risk of African trypanosomiasis (AT), caused by tsetse fly-transmitted protozoan blood parasites of the Trypanosoma genus. Animal African trypanosomiasis (AAT), or nagana, is caused by T. brucei, T. congolense and T. vivax and negatively impacts livestock farming and consequently the economy of the continent. Since AAT occurs in rural areas, affordable rapid diagnostic tests (RDTs) and drugs are required. Diagnostic tests focus on antibody detection; however, antigen detection is more favorable since anti-trypanosome antibodies persist in blood for years following recovery. Due to the parasite’s defense by antigenic variation, development of a vaccine is unlikely. Molecules that are essential for parasite survival, such as peptidases, are currently being targeted for diagnosis and chemotherapy. A cathepsin-L-like cysteine protease from T. vivax, TviCATL, is released by dying parasites in the host bloodstream and was shown to be a diagnostic target for detecting host antibodies. To achieve diagnosis of current infections, detection of TviCATL is being explored. The overall aim of this study was to enzymatically characterise TviCATL; and to study the interaction of antibodies against the TviCATL antigen which could be used as a chemotherapeutic drug for the diagnosis of T. vivax infections. The protease, TviCATL, was recombinantly expressed in E. coli using the pET-28a expression vector and purified using a nickel chelate affinity column. The resulting 47 kDa protein was identified using western blot and was shown to hydrolyse H-D-Ala-Leu-Lys-AMC and was inhibited by bestatin and E-64 and had optimal activity between pH 6.5 and 7.5. The crossreactivity between TviCATL and antibodies produced against other Trypanosoma spp cysteine proteases was evaluated in western blots, and results confirmed cross-reactivity. In addition, chicken anti-TviCATL antibodies were able to detect TviCATL in TviCATL-spiked bovine serum. The production of antibodies using the Nkuku® phage library was employed as an alternative to the animal-based antibody production and single-chain variable fragment (scFvs) antibodies were selected by panning against the TviCATL antigen. After four rounds of panning, TviCATL-scFvs binders were enriched and four clones gave the highest signal when evaluated using a monospecific ELISA. Due to the low values obtained, optimisation of panning is necessary for improved results. Optimisation of recombinant expression and purification of the identified scFvs for use in a sandwich ELISA were explored to this end. This study showed that TviCATL is a promising chemotherapeutic and diagnostic target for African animal trypanosomiasis.
The role of MMP-14 and MMP-2 in mediating myoblast fusion.
(2016) Nkosi, Mthokozisi Siphesihle.; Niesler, Carola Ulrike.
Satellite cells are muscle precursor cells that have the ability to self-renew, proliferate and differentiate into myoblasts that eventually elongate and fuse to form myotubes which are vital for regeneration and repair of muscle. Satellite cells reside in a niche, between the sarcolemma of the muscle fiber and the basal lamina, which consists of mostly collagen IV, proteoglycans and laminin. Matrigel is a gelatinous protein mixture that consists primarily of collagen IV and laminin and therefore resembles the basal lamina. Matrix Metalloproteinases (MMPs) are zinc endopeptidases, proteolytic peptidases which break peptide bonds within their substrates. MMP-14 (membrane bound) also known as membrane-type 1 matrix metalloproteinase (MT1-MMP) is one of the major matrix metalloproteinases (MMPs) involved in muscle repair and regeneration, together with MMP-2 (secreted). MMP-2 is a secreted gelatinase A, which is activated by MMP-14. MMP-2 is also known to be activated by nitric oxide (NO), therefore allowing active MMP-2 to release growth factors such as Hepatocyte Growth Factor (HGF) from the extracellular matrix (ECM). There are two forms of MMP-2, intracellular MMP-2 and extracellular (secreted) MMP-2. Secreted MMP-2 contains a peptide signal that helps direct it outside the cell, while intracellular MMP-2 lacks this feature and is therefore retained within the cell. Intracellular MMP-2 activity is known to be a major cause of muscular atrophy. Secreted MMP-2 is known to degrade ECM components, facilitating satellite cell mobility and release of growth factors such as HGF, aiding in muscle regeneration. MMP-2 can cleave collagen IV due to the presence of a fibronectin-like domain within its catalytic domain; this is not the case with MMP-14. MMP-14 and MMP-2 together degrade collagens, fibronectin, laminin-2/4 and other adhesion molecules. This clears the path for the myoblast to align and fuse to form myotubes which then finally align to form mature muscle fibers. The levels of MMP-14 and MMP-2 must be regulated; low levels can cause muscular dystrophy. The current study analysed expression levels, activity and role of MMP-14 and MMP-2 in C2C12 myoblast differentiation. C2C12 myoblasts first proliferated (Day 0), then aligned and elongated (Days 1-2) and then finally fused into myotubes (Days 3-5) during differentiation. MMP-14 and MMP-2 protein levels were high during the elongation period and also during fusion of C2C12 myoblasts. MMP-14 was localised at the focal adhesions, where actin filaments terminate during myoblast proliferation and fusion. ii Inhibition of MMPs using BB94 (10 µM) was observed to significantly reduce C2C12 myoblasts fusion. Secreted MMP-2 seems to play a vital role in the C2C12 differentiation, as activity was seen during myogenesis; when neutralised with an antibody, an 18% decrease in fusion was observed. Matrigel promoted an increase of MMP-2 expression within the cell during fusion (day 5 of differentiation), while no intracellular MMP-2 protein was observed at day 2 of differentiation. Levels of secreted MMP-2 increased significantly from day 2 to day 5 of differentiation; however, the presence of Matrigel significantly reduced levels of secreted MMP-2 detected in conditioned media at day 5 compared to uncoated conditions. The decrease is, in part, due to the fact that MMP-2 was found to bind to Matrigel. In conclusion, MMP-14 and MMP-2 play an important role in C2C12 myoblast elongation and fusion. This study provides further insight into the role of MMPs in myogenesis and lays the foundation for future work.
Intercellular communication between fibroblast phenotypes, macrophages and myoblasts during cellular migration.
(2022) Ramklowan, Dhamini Sanjay Hariduth.; Niesler, Carola Ulrike.
Abstract available in PDF.
Identifying small molecule binding ligands for the pup-ligase (PafA) of Mycobacterium tuberculosis.
(2022) Mthembu, Sandile Mehzi.; Hewer, Raymond.; Delport, Alexandre' Marie Chaplin.
The rapid emergence of resistant TB strains (Mycobacterium tuberculosis (Mtb)) renders traditional treatment options ineffective and necessitates the generation of novel anti-TB drugs that possess innovative modes of action. The pup-ligase (PafA) of Mtb that solely mediates protein proteasomal removal via the pupylation cascade has recently been identified as a suitable target for TB drug development. A novel approach would be to recruit proteolysis targeting chimeras (PROTACs) technology as an alternative anti-TB treatment option by developing PROTAC-like molecules capable of recruiting the pupylation cascade. Therefore, the identification of novel PafA small-molecule binding ligands is an essential first step to establish possible new TB therapies. To this effect, PafA recombinant expression was successfully optimised in E. coli cells at 20°C for 20 h, where a 50-kDa protein was observed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the identity of the protein was confirmed via immunoblotting with anti-His antibodies and PafA subsequently purified via immobilized metal affinity chromatography (IMAC) to high purity. A thermal shift assay (TSA) of PafA against 48 small-molecule compounds from a chemical library pre-screened for non-specific inhibition activity was conducted. Seven Hit compounds were detected significantly binding PafA (P < 0.05), all inducing a > 5 °C increase of PafA melting temperature (Tm) upon binding. Future research on these novel PafA binding ligands will be to ascertain whether they possess inhibitor qualities. Additionally, they will be used in the synthesis of heterobifunctional molecules to create the first PROTAC-like molecules for targeted proteasomal degradation of essential Mtb proteins – a novel type of anti-TB drug.
Investigation of intravaginal practices as a factor associated with a high prevalence of genital human papillomavirus infection in adolescent girls.
(2021) Mntambo, Ntombenhle.; Gumbi, Pamela Phumelele.; Ngcapu, Sinaye.
Background: Human papillomavirus (HPV) is a common sexually transmitted infection (STI) in women which mainly infects the mucosal areas. Young women are disproportionately infected by HPV, and factors that may render adolescents or younger women more vulnerable to HPV acquisition than older women have not been fully elucidated. This study aimed to investigate the associations between the prevalence of HPV, the use of intravaginal products, the immune activation status of cervical T cells, and alterations of the composition and concentrations of antimicrobial peptides (AMPs) in vaginal fluid among adolescent females close to their sexual debut and older women in KwaZulu-Natal. Methodology: Genital specimens (cervical cytobrush and cervicovaginal swabs) were collected from 154 female participants aged 14-19 and 25-35 years. From cervicovaginal swabs, HPV genotyping was done using a deoxyribonucleic acid (DNA) Flow hybridization system. Flow cytometry was conducted from cervical cytobrush specimens (evaluating CD38+, HLA-DR+, andCCR5+ expressions) to assess T-cell immune activation status. Enzyme-linked Immunosorbent Assay (ELISA) kits were used to measure genital concentrations of human β- defensin (HBD-1, HBD-2), and psoriasin from cervicovaginal swabs. Statistical tests conducted were Robust Poisson regression models (RPRM), the Tukey multiple comparison adjustment, t-test, and Mann Whitney U test. 𝑃 values of <0.05 were statistically significant. Results: HPV prevalence was 85%, with high-risk genotypes being the most prevalent. The majority of the cohort was infected with multiple genotypes (76.62%). Genotypes associated with cancer and current Gardasil®9 HPV vaccine targets were more common (53.9%) than those associated with genital warts (14.9%). The risk of HPV in adolescent females was 15.9% higher than in adult females. The use of vaginal inserted products (VIPs) was associated with a 40% higher risk of contracting genotypes related to cancer (p=0.0503) compared to nonusers. The risk of HPV infection for adolescents using VIPs was 23% higher than that of adults using VIPs. However, these differences were not statistically significant at a 5% level of significance. Sexual debut after 18 years significantly reduced the risk of overall HPV infection (p=0.0040) and infection with genotypes associated with cancer (p=0.0024). When comparing HPV infected adolescents and adults, the proportion of activated CD4+ T cells was significantly higher in adolescents, particularly in CD4+ HLA-DR+ cells (p=0.0008). CD8+ T cells showed no difference. A significantly higher concentration of HBD-2 was observed in HPV+ adults compared to HPV- adults (p=0.0215) and HPV+ adolescents (p=0.0189). Conclusion: The overall HPV prevalence is higher than the previously reported prevalence in KwaZulu-Natal province. In addition, we demonstrate that the use of VIPs may be associated with some HPV infection risk, particularly in adolescent females. This finding suggests that young women should be warned about the potential risk of using VIPs. We also confirm that the age-phase, delay in sexual debut, and the number of sexual life partners have significant associations with HPV genotypes linked to cancer, highlighting the importance and the urgency of vaccinating young girls with Gardasil®9 HPV vaccine. Adolescents with HPV have significantly higher levels of activated CD4+ T cells in their cervical mucosa, suggesting the presence of reactive activated cells that lack efficiency in the clearing of HPV infection in this age group. HPV infection upregulates HBD-2 levels during HPV infection, notably significantly higher in adult females than in adolescents. This investigation has generated new insights into the risk factors for HPV acquisition in young women. These findings need to be confirmed further by larger cohort size studies, essential for HPV prevention.
Biochemical and structural characterization of ClpK from Klebsiella pneumoniae.
(2022) Motiwala, Tehrim .; Khoza, Thandeka.
Abstract available in PDF.
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.
Identification and analysis of Cryptosporidium Glutathione Transferase.
(2021) Mfeka, Sizamile Mbalenhle.; Khoza., Thandeka Ntokozo.
Cryptosporidiosis, caused by Cryptosporidium spp. is a gastrointestinal disease which gives rise to severe life threatening complications in immunecompromised patients. The disease causing parasite has a proficient defense system against xenobiotic compounds and substances that renders the only drug designed to treat the gastroenteritis infection inefficient in immune compromised patients. This defense system includes a phase II enzyme called Glutathione Transferase (GST) which detoxifies a wide range of oxidant based substrates. The overexpression of this protein in multi drug resistant cases and its presence in multiple stages of the parasites life cycle highlights the parasites dependence and utilization of the GST protein thus making it a suitable therapeutic target. This study was then set out to determine characteristic features of Cryptosporidium GSTs in comparison to well studied GSTs using molecular biology and bioinformatics tools. A genome wide search was performed across multiple protein databases to mine the Cryptosporidium GST. The 15 Cryptosporidium spp. found to possess full length proteins were compared amongst themselves within the species and against other species using phylogenetic analyses. This led to the discovery of three novel classes of Cryptosporidium GST based on amino acid sequence identity. The classes were named Gamma, Psi and Vega GSTs. The GSTs varied in amino acid length, and secondary structure characteristics determined through homology modeling. In comparison to preexisting GSTs, the Psi and Vega class GSTs did not have the typical active site Tyr7 found in most cytosolic GST, furthermore the Vega class GST also did not have the typical thioredoxin like fold conserved in the N-terminal region of all GSTs. The Gamma class GSTs were found to most resemble pre existing GSTs consisting of the typical thioredoxin fold and the active site Tyr7 and thus selected for expression and purification studies. pET, pCOLD1 and pCOLDTF vectors were used to determine a suitable vector to facilitate the expression of a soluble gamma class GST in Escherichia coli. pCOLDTF which utilizes cold shock proteins at low temperatures and a chaperone called trigger factor assisted in the recombinant expression of the gamma class GST resulting in a protein with the monomer size of ~50 kDa, which is double that of existing GSTs. This is owed to by the N-terminal and C-terminal extensions that the protein possesses. The protein was purified to homogeneity using affinity chromatography and size exclusion chromatography. The resulting protein was found to be dimeric under native conditions.

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