Theses and Dissertations (Biochemistry)

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The Antiatherogenic Potential of Two triterpenes from Protorhus longifolia stem bark (Benrh.) Engl.
(University of Pretoria, 2024-01) Mosa, Rebamang; naleli.ramasia@gmail.com; Ramasia, Naleli Nthabiseng
Atherocardiovascular diseases (ACVDs) are considered among the major causes of death in South Africa and worldwide. Hypercholesterolemia, which is the main major risk factor of atherosclerosis, increases the occurrence of ACVDs. Despite the use of the current drugs to treat ACVDs, the continuous increase in morbidity and mortality rates associated with atherocardiovascular events indicate limitations of the current treatment regime. Medicinal plants are known to contain numerous compounds with various bioactivities including antioxidant capacity and lipid lowering properties. The triterpenoid compounds (ARM-2 and RA-5) isolated from Protorhus longifolia (Benrh.) Engl. stem bark have shown promising bioactivities such as inhibiting dietary cholesterol digestion and potential to regulate the endogenous pathway of cholesterol biosynthesis. These properties play a vital part in the amelioration of hypercholesterolemia. This study investigated the antiatherogenic potential of ARM-2 and RA-5. The computational and wet lab experiments were implemented to investigate the preventative properties of the compounds against oxidative modification of LDL and foam cell formation that underlie the progression of atherosclerotic plaque. The in silico molecular docking of the compounds against some proteins involved in cholesterol trafficking, and their ADMET properties were determined. In vitro analysis of potential bioactivities of the compounds was determined through antioxidant assays (ABTS, DPPH, ferric reducing power and ORAC), and inhibition of the CuSO4 mediated LDL oxidation. The oxidized LDL-induced foam cell formation experiment was carried out to determine the inhibitory activity of the compounds against foam cell formation in RAW 264.7 cells. Molecular docking of the compounds indicated poor binding affinity against CD36, SR-A1, ABCA-1 and sPLA2 in contrast to strong binding affinities against ABCG-1, ACAT-1 and LOX-1. Both compounds were predicted to have promising ADMET properties and drug-likeness depicted by high intestinal absorption and no inhibition observed for the cytochrome P450 isoform. The triterpenes showed poor free radical scavenging activity (ABTS and DPPH) and reducing power but indicated good activity against peroxyl free radicals. Inhibition of oxidative modification of LDL was observed by decreased CD and MDA formation by both compounds at 500 μM. However, the compounds were unable to inhibit sPLA2 activity. Except for RA-5 at 25 μM, high viability of the cells was observed following their exposure to both triterpenes. The compounds decreased intracellular lipid droplet formation, ultimately resulting in decreased foam cell formation by 14% and 10% for ARM-2 (25 μM) and RA-5 (10 μM) respectively. Overall, the results from the study revealed that both triterpenes possess bioactive properties to inhibit oxidative modification of LDL and decrease foam cell formation, highlighting their antiatherogenic potential.
Inhibiting amylolytic enzymes by both acarbose and cellobiose as a prelude to treating type II diabetes mellitus
(University of Pretoria, 2024-02-14) Malgas, Malgas; Malgas, Samkelo; u18200941@tuks.co.za; Maluleke, Kamogelo C
Inhibition of starch-degrading enzymes, α-amylase and α-glucosidase, provides a measure to ameliorate type II diabetes mellitus (T2D) by limiting the amount of glucose produced from dietary starch that would subsequently be absorbed into the bloodstream. This study investigated the inhibitory potential of cellobiose in amylolytic enzymes alone and also assessed its synergistic effects when combined with the gold AGI standard, acarbose. Firstly, the pharmacokinetic properties prediction and gastrointestinal digestibility simulation of cellobiose were investigated. Following, in silico molecular docking, in vitro enzyme inhibition, and UV spectroscopy were then used to investigate the inhibitory potential of cellobiose. Lastly, single and combined acarbose and cellobiose were investigated for their inhibition of the amylolytic enzyme cocktail. Cellobiose showed drug-likeness properties and did not possess any toxicity. In addition, it was found to remain stable under gastrointestinal simulated conditions. Acarbose (-7.3 kcal/mol and -8.2 kcal/mol) had the highest binding affinity than cellobiose (-6.0 kcal/mol and -7.5 kcal/mol) for both α-amylase and α-glucosidase, respectively. Upon binding of the compounds to the targets in vitro, acarbose (Ki = 0.012 mM) is a reversible uncompetitive inhibitor, while cellobiose (Ki = 2.2 mM) is a reversible non-competitive inhibitor of α-amylase. On the other hand, both acarbose (Ki = 0.08 mM) and cellobiose (Ki = 14 mM) are reversible competitive inhibitors of α-glucosidase. A combination of acarbose and cellobiose in different ratios resulted in more synergistic results than antagonistic or additive effects, with a 0.005: 1.25 mM (acarbose: cellobiose) being the best combination. The results of the study showed that although cellobiose is not a better inhibitor of amylolytic enzymes, its combination with acarbose leads to synergism which may reduce side effects presented by the gold AGI standard and, as a result, both have the potential to be used for T2D treatment.
Exploring the mode of action of MMV1580843 against Plasmodium falciparum
(University of Pretoria, 2023-12-12) van der Watt, Mariëtte Jr; Birkholtz, Lyn-Marie; u17031983@tuks.co.za; Schultz, Dana Caitlin Jr
Although malaria is both preventable and curable, it is a parasitic disease with the highest fatality rate in history, causing over half a million deaths worldwide each year, with 95 % of the cases occurring in Africa. Female Anopheles mosquitoes act as vectors for the malaria parasite. Plasmodium is transmitted to a human host when a female Anopheles mosquito feeds on the individual. In turn, humans infected with the malaria parasite can transmit the parasite back to another mosquito, perpetuating the parasite’s life cycle. The presence of both insecticide and therapeutic drug resistance to current antimalarials is an ongoing threat. Therefore, it is crucial that the next generation of antimalarials include compounds with unique modes of action and exhibiting high barriers to resistance development. Blocking transmission is essential for malaria elimination, given that current drugs primarily target asexual parasites or sporozoites and liver stages. The lower number and non-replicating nature of gametocytes render the development of resistance to transmission-blocking antimalarials highly improbable. Gametocyte-specific antimalarials are presumed to target unique biological processes compared to compounds targeting asexual parasites, reflecting the fundamental difference in the biology associated with asexual proliferation and gametocyte differentiation. A key drawback of phenotypic screening of candidate antimalarials is the lack of knowledge regarding the mode of action of a compound, which is the bottleneck of drug discovery and development. While current target identification strategies predominantly focus on the asexual stages of the parasite life cycle, there is a noticeable gap in knowledge regarding the mode of action of gametocyte-specific antimalarials. MMV1580843, a unique chemotype, is a structurally diverse and potent transmission-blocking compound, showing stage-specific activity towards non-proliferative gametocytes. This TCP-5 (target candidate profile-5, transmission-blocking) selective compound, with a potential novel mode of action in Plasmodium, was investigated here. This project thus aimed to determine the mode of action of MMV1580843 against P. falciparum late-stage gametocytes through biochemical approaches. The findings presented in this study point towards the mitochondria of late-stage gametocytes being the target of MMV1580843, supported by investigations involving fluorescent indicators and cross-reactivity with antimalarials having confirmed modes of action. The results of this research exhibit a promising step forward in elucidating the mode of action of a gametocyte-specific antimalarial, providing a foundation for the development of targeted interventions to disrupt malaria transmission. By bridging the existing gap in knowledge and proposing an innovative strategy, this study contributes to the ongoing efforts in the global fight against malaria.
Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum
(University of Pretoria, 2023-12-14) Birkholtz, Lyn-Marie; Niemand, Jandeli; u18001638@tuks.co.za; Van Zyl, Christea
Malaria cases have surged in recent years, with the World Health Organization reporting 619 000 deaths in 2021. A recently approved malaria vaccine has been recommended for children younger than 5 years; this vaccine, however, does not prevent cases amongst older children and adults. This stresses the necessity to allocate resources wisely for sustainable malaria control and treatment, emphasized by the ongoing threat of antimalarial resistance. Advancements in knowledge of the most lethal malaria-causing species, Plasmodium falciparum, offer hope for malaria elimination. P. falciparum genetic modification strategies have been a growing field, allowing for the targeting of specific components of this deadly parasite's genome. Controlled genetic interrogation at specific time-points within the highly dynamic and complex life cycle of the parasite has become an increasingly popular method for establishing the essentiality of genes in this parasite. However, non-inducible genetic knockout (KO) interrogation strategies do not facilitate the genetic probing of regulatory genes, due to the immediate death phenotype observed. The Dimerisable Cre recombinase (DiCre) system is a KO genetic interrogation tool used to flox any nucleotide sequence flanked with the cre-specific loxP sites, but only when induced with rapamycin. A drawback of this system is the current cloning approach employed for this system, which relies on continuous cloning of selection markers used for screening, and these critical loxP sites. This highlights the demand to bridge the gap and establish a more regulated cloning approach to generate transgenic parasite lines. In this study, we developed a new DiCre cloning strategy. Specifically, we created a universal DiCre repair plasmid containing these loxP sites, together with a selection marker cassette and multiple cloning sites for homology regions and a recodonised gene insert. This will facilitate genomic integration through either the CRISPR-Cas9 or SLI genetic modification systems. The application of this technology was explored using two proxy regulatory genes, gcn5 and set7, which play a role in histone post-translational modifications across the parasite's epigenome. This study presents a unique cloning approach for conditional gene knockout and supports future research to help expand our knowledge of gene essentiality in the P. falciparum parasite.
Antibiofilm properties of selected Lactobacillus species and their mechanism of action against Listeria monocytogenes
(University of Pretoria, 2022-01) Thantsha, Mapitsi Silvester; u14008794@tuks.co.za; Masebe, Reabetswe Dolly
The disastrous effects of the foodborne pathogen Listeria monocytogenes have been observed overtime by global listeriosis outbreaks claiming many lives. The spread of this pathogen is being reported at high levels with persistence spanning over several years in food-processing environments. The main source of contamination occurs in these very environments where L. monocytogenes present on surfaces comes into contact with food products and later infect consumers. The situation is exarcebated by the fact that L. monocytogenes forms biofilms, one very powerful virulence mechanism, that adhere to processing surfaces. The shortfall of commonly used antibiotics and sanitizers as treatment against L. monocytogenes biofilms due to antimicrobial resistance leads to a major safety crisis within the food industry. This limitation of antimicrobial agents sparked an interest in research of lactic acid bacteria (LAB) with certain probiotic properties as an alternative antibiofilm agent. Research has highlighted that the use of substances produced by LAB achieves inhibition and dispersal of L. monocytogenes biofilms. The antimicrobial substances of LAB have been revealed to showcase antilisterial activity, contributing to the combat of biofilm formation on surfaces. It is however known that the properties of probiotics cannot be generalized. Taking these into consideration the current study aimed to determine whether selected Lactobacilli had the potential as an alternative control measure for biofilms formed by L. monocytogenes isolated from the food or food environments. The initial step of the current study was to screen L. monocytogenes strains for biofilm formation abilities. Then subsequently, the capabilities of cell free supernatant (CFS) of selected LAB strains (Lactobacillus acidophilus La14 150B, Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus ATCC 4356) to inhibit as well as disperse the formation of biofilms of L. monocytogenes strains was investigated. L. monocytogenes ATCC 19115 and L. monocytogenes Cuc (originally isolated from cucumber) were classified as moderate biofilm formers, while L. monocytogenes 243 and L. monocytogenes Avo (isolated from avocado) were classified as strong biofilm formers. The strains isolated from the food processing environments were better biofilm formers that than the positive control L. monocytogenes ATCC 19115. After determining these categorizations, the L. monocytogenes strains were treated with CFS of LAB. All CFS managed to inhibit the formation of biofilms across all strains, significantly decreasing their biofilm former categories from either moderate or strong to weak (p < 0.05). L. acidophilus La14 150B performed better overall in inhibiting the biofilms. Cocktails of the CFS of LAB were prepared and additionally used as treatment. The cocktail ABC (L. acidophilus La14 150B + L. plantarum + L. rhamnosus ATCC 4356) had more efficient inhibition capabilities with both L. monocytogenes Avo and L. monocytogenes Cuc having the lowest recorded optical density values post treatment. The following step in this experiment explored the dispersal capabilities of the CFS of LAB. Though the preformed biofilms were not completely dispersed following treatment, the CFS were able to change the classification of all the L. monocytogenes strains into weaker biofilm former categories. L. acidophilus La14 150B was the most efficient of all LAB in removal of biofilms with significantly reduced optical density values (p < 0.05). Then, different cocktails of the CFS were prepared and assessed for their biofilm removal capabilities. It was deduced that all three CFS of LAB were able to significantly disperse the biofilms (p < 0.05). Collectively, the cocktail ABC (containing CFS of the three LAB test strains) had the greatest efficiency in dispersal abilities. From these results it was evident that cocktails were superior to individual CFS in the inhibition and dispersal of L. monocytogenes biofilms. The observed inhibition and dispersal abilities of CFS of LAB (L. acidophilus La14 150B, L. plantarum and L. rhamnosus ATCC 4356) raised an interest in seeking to investigate their potency in removing L. monocytogenes biofilms adhered to different surfaces similar to those in contact with food in food processing environments. Scanning electron microscopy (SEM) revealed that both L. monocytogenes ATCC 19115 and L. monocytogenes 243 have a different attachment style on the hydrophilic stainless steel in contrast to the hydrophobic polyvinyl chloride (PVC). A two-layered biofilm structure with a honey comb complex was observed for both these L. monocytogenes strains on stainless steel which was noticeably absent on PVC. Moreover, for the removal of L. monocytogenes biofilms on stainless steel coupons by CFS of LAB, all treatments managed to disperse the aggregated structures resulting in isolated cells. The antibiofilm properties of the different LAB was evidenced by a decline in the cell-clusters of L. monocytogenes ATCC 19115 and L. monocytogenes 243. On PVC, the three CFS exhibited the ability to interfere with and disrupt the aggregation of L. monocytogenes cells to each other as scattered cells were observed post treatment. Overall, L. acidophilus La14 150B dispersed the L. monocytogenes ATCC 19115 and L. monocytogenes 243 biofilms on both the stainless steel and PVC surfaces with the highest efficiency. In order to determine the mechanism by which CFS of LAB interfered with the L. monocytogenes biofilm formation capabilities, the study further investigated how expression of the L. monocytogenes prfA gene is affected by the presence of CFS of LAB. The results demonstrated that the presence of all CFS of LAB (L. acidophilus La14 150B, L. plantarum and L. rhamnosus ATCC 4356) caused a significant downregulation in the expression of prfA (p < 0.05). This downregulation affects the coding of the PrfA regulator protein and disrupts L. monocytogenes biofilm formation. In correlation with the results observed for SEM analysis, the CFS of L. acidophilus La14 150B exhibited the highest antagonistic behavior, with expression of prfA recorded at a low 23% after treatment. The results of the present study suggest that the CFS of LAB contain specific substances with antilisterial activity and antibiofilm properties. The presence of these substances contained within the CFS of LAB negatively affects the virulence gene prfA, decreasing the ability of L. monocytogenes to form biofilms. The study is of importance to the various food processing facilities and the food industry as it provides a potential safe alternative that can be used to limit any further outbreaks due to contamination of food products by L. monocytogenes. Cell free supernatants with demonstrated antibiofilm properties could be incorporated in industries and stipulated as the standard control measure for L. monocytogenes biofilms. Furthermore, for an enhanced effective treatment the cocktails of such CFS could be very beneficial. Thus cell free supernatants of lactic acid bacteria can be used to curb the formation of L. monocytogenes biofilms on food processing surfaces in the food industry and thereby contribute to improved food safety.
Non-compliance assessment and law enforcement efficacy in the eMalahleni Local Municipality Mpumalanga
(University of Pretoria, 2021) Chirwa, Evans M.N.; vonganicory@gmail.com; Ngobeni, Vongani
Compliance refers to complying with legislation and/or licensing requirements, while regulation refers to the use of legislative powers to ensure compliance with the law (commercial law, 2016). It may also entail measures to ensure that companies comply with both industry and government regulations. Wastewater treatment works are required to comply with the operational standards of Green drop requirements and regulations from the Department of Water and Sanitation and are advocated by the National water Act, (Act 36 of 1998). As the highest law in the country, the Constitution of South Africa (Act 108 of 1996) represents the most overarching legal framework for wastewater services regulation. Where the Constitution, together with the Municipal Structures Act (Act 117 of 1998) and the Water Services Act (Act 108 of 1997), assign the responsibility for the provision of wastewater services to the local sphere of government (municipalities). In this regard some municipalities have been classified as WSAs that are responsible for providing water services within their areas of jurisdiction (RSA, 1997). This study pursue to assess the non-compliance effluent and impact of partially treated effluent discharged into water resource and the environment from wastewater treatment plants in eMalahleni local municipality, identifying challenges and risk. This was achieved through cross section study using qualitative data collection method. Data was collected through previous National Green drop assessments conducted by the Department of Water and Sanitation, resident’s interviews and local newspapers as well with physical operational plant assessment. Three questionnaires concerning the understanding and identification of the cause and impact of the discharged non-compliance effluent into a water resources and environment was investigated; (a) challenges leading to non-compliance of effluent (b) the impact of non-compliance effluent on environment (c) law enforcement challenges in water and wastewater management. Overall, the findings revealed that the two study wastewater treatment works operation is not satisfactory, they are not fully applying nor implementing the operational requirements of wastewater as stipulated by the regulator department and not implementing all the recommendations provided during National Green drop assessments conducted on this Municipality. There is an indication of plants operated by unskilled personnel who still require training on operations of wastewater treatment works. Both plants were found to be at high risk, do not comply with Ammonia and was concluded that will be placed under surveillance due to possibility of posing a health threats to the surroundings. The Municipality tap drinking water was no longer safe to drink and had a brown colour, it was found that the residents were not pleased with the water situation in eMalahleni. Local business were affected, guest at Hotels were told not to drink water from the taps. Challenges included a lack of commitment from management, attitude of operators, poor maintenance and budget, lack of an iii updated wastewater risk abetment plan, process audits and asset management, as well as politics and lack of public awareness. In conclusion, there are measures and regulations in place that can solve the noncompliance effluent challenges and increase compliance. This involves, among other things, ensuring compliance with the country’s water legislation, particularly water quality requirements, and taking enforcement actions in cases of non-compliance (DWAF, 2003; Ntombela, 2013), however they were ignored. It is concluded that all findings contributed to unsafe drinking water in eMalahleni. In light of these research findings, it is recommended that all Green drop requirements measures outlined be implemented so that compliance can be achieved. If this is not successful, the relevant DWS regional office will issue a directive, and if the conditions of the directive are not adhered to criminal charges may be laid against the municipality (DWA, 2010).
Chemically synthetic mycolic acids as vaccine adjuvants
(University of Pretoria, 2014) Verschoor, J.A. (Jan Adrianus), 1953-; Lemmer, Yolandy; Heena_Ranchod@yahoo.com; Ranchod, Heena
No abstract
Development and evaluation of a bi-enzymatic nitric oxide reduction system
(University of Pretoria, 2013) Verschoor, J.A. (Jan Adrianus), 1953-; Jordaan, Justin J.; seikegarny@gmail.com; Garny, Seike
Nitric oxide is a small diatomic molecule and is part of the nitrogen radical species. As a gas, it diffuses easily across cell membranes and is involved in numerous physiological processes and inflammation. This peculiar molecule has a dual role in inflammation. NO is one of the first signals to commence an innate immune response and it is involved in the resolution of inflammation. In the control of inflammation it is crucial to resolve NO bursts to promote tissue healing. The failure thereof results in the progression of inflammation with potentially catastrophic consequences for the host. This study aimed to develop a nitric oxide reduction system as a research tool which could facilitate the understanding of the intricate role of NO in inflammation. Numerous chemical tools have been used to study NO biology, but were found to interfere in other metabolic pathways; thereby masking the role of NO. The nitric oxide reduction system entails the reduction of NO by nitric oxide reductase (NOR) with the concomitant oxidation of glucose by glucose dehydrogenase (GDH). The latter enzyme recycles the cofactor NADH in such a way that NO is continuously reduced. This bi-enzymatic cofactor recycling system presents the advantage of NO removal without any interference of metabolic pathways. Here, we propose that the continuous reduction of NO by the NOR system could be used to elucidate the role of NO in an innate immune response. The construction of the NOR system commenced with development of fast and reliable spectrophotometric NADH-enzyme activity assay. This assay was essential for the quantification of enzyme activity and was used throughout the study for the purification of NOR, characterisation of NOR as well as the determination of enzyme activity maintenance after enzyme immobilisation. Both enzymes were immobilised onto five carriers with two different functional group chemistries and three functional group densities. The carboxyl functionalised carrier with the lowest functional group density was the most suitable immobilisation carrier by maintaining the highest enzyme activity for NOR and GDH. Upon co-immobilisation of both enzymes, an average of 0.088 μmoles NADH.min-1 for NOR and 0.077 μmoles NAD.min-1 for GDH cofactor oxidation rate was achieved. Furthermore, the cofactor was recycled six times with the concomitant consumption of the enzymes’ substrates. Subsequently, the NOR system was evaluated for its potential as a research tool in an in vitro inflammation model. The continuous reduction of NO was established which highlights the NOR system suitability as a research tool. However, its evaluation as a potential anti-inflammatory reagent indicated that the chosen carrier has immunogenic properties of its own. The inflammation response elicited by this carrier alone was in part abrogated by the immobilisation of enzymes in the eventual NOR system assembly, thereby providing a scope for future work and further optimisation of this anti-inflammatory reagent.
Transcriptional regulation underlying the quantitative genetic response of maize to grey leaf spot disease
(University of Pretoria, 2014) Berger, David Kenneth; Myburg, Zander; nanette.coetzer@gmail.com; Christie, Nanette
Cercospora zeina causes grey leaf spot (GLS), a yield-limiting disease on maize. The main objective of this study was to exploit maize gene expression data to dissect the quantitative disease response to C. zeina infection. The project addresses the hypothesis that there is an underlying DNA polymorphism that gives rise to a change in gene expression, which in turn affects GLS disease severity. Genomic and functional annotation of the reporters on an Agilent 44K maize microarray was carried out. This microarray was used for global gene expression profiling of earleaf samples collected from 100 recombinant inbred sub-tropical maize lines exposed in the field to C. zeina. Gene expression profiles together with GLS severity scores were used in a weighted gene co-expression network analysis to identify co-expression modules associated with disease severity. Quantitative trait locus (QTL) mapping for GLS severity was combined with expression QTL (eQTL) analyses to investigate the molecular basis of the quantitative response to GLS. An Eqtl data analysis pipeline was developed in Galaxy. The overlap of phenotypic QTLs with cis- and trans-eQTLs revealed putative causal candidate genes and potential mechanisms responsible for the QTLs, respectively. Regulatory network models were constructed for trans-eQTL hotspots coinciding with phenotypic QTLs. A genetic basis for coordinated expression responses to GLS disease was identified. For the susceptible response, the results lead to the hypothesis that a calmodulin-related protein with a cis-eQTL acts as a global regulator of various pathogenesis-related proteins that are activated too late after infection started. For the resistant response, it is hypothesised that a serine threonineprotein kinase with a cis-eQTL acts as a post-translational global regulator regulating phosphatases and kinases involved in activation of defense gene expression. The outcomes of this study were: i) the development of a systems genetics strategy and ii) several hypotheses of maize transcriptional responses to C. zeina which need to be validated with further studies. These results extend the current knowledge of GLS resistance and could aid in the improvement of maize varieties.
Basal promoter landscape in Eucalyptus grandis : annotation of distal transcription start sites and core promoter usage
(University of Pretoria, 2014) Myburg, Alexander Andrew; Mizrachi, Eshchar; Joubert, Fourie; ida.vanjaarsveld@gmail.com; Van Jaarsveld, Ida CecIlia
Transcription is a complex biological phenomenon, whereby RNA is transcribed from single stranded template DNA by assembling targeted regulatory inputs at the promoter region. Transcription is regulated through many hierarchically organised mechanisms, including chromosome positioning and organisation, the binding of transcription factors, and DNA’s secondary and tertiary structures at the region of transcription initiation. The core promoter is the distinct functional unit of DNA overlapping the transcription start site, which possesses linear regulator capacity and renders DNA permissive to transcription. In plants, core promoter and enhancer studies are of particularly high impact for those traits which under strong transcriptional control. Cellulose biosynthesis in immature xylem, the tissue which forms wood, is one such trait, and is studied extensively in the herbaceous model plant organism, Arabidopsis thaliana, and the economically important woody perennial, Eucalyptus grandis. The release of the E. grandis genome sequence has provided a muchneeded reference to study transcriptional control, not only for those traits that make it a dominant fibre crop, but genome-wide. We aimed to use empirical transcript evidence to perform a high-throughput genome-wide curation of the 5’ UTR annotations and empirically infer transcription start sites (TSSs) of the nascent E. grandis genome annotation. We then aimed to use the curated TSSs to define core promoter classes based on their sequence Magister composition and to determine the putative expression profiles and functional associations of each. We used deep E. grandis mRNA sequencing data across seven diverse tissues and PASA assembled E. grandis ESTs to empirically curate 5’ UTR annotations. We improved 17,085 annotations, added 7,596 for which there was no previous annotation and retained 3,675 that possessed only a predicted TSS without empirical evidence. These complementary data were used to define distal transcription start sites (dTSS) by a novel, prioritising, computational rule-based method. From these dTSS annotations, we extracted the core promoters (from -100 to +50) and described the core promoter landscape by hexamer positional overrepresentation analysis. We found three types of hexamer over-representation in the core promoter, that being broad, spiked and low. Broad hexamers were classified into 5 distinct core promoter classes, including TA, CT, GA, W and S. These were further assessed for putative expression profiles (specificity and level) and functional associations. TA resembles the conserved TATA-box core promoter, although displays a bimodal distribution, low expression levels and the greatest tissue specificity. CT and GA are over-represented both up and downstream of the dTSS and show narrow windows of greater enrichment with phasic constraint. W and S occur in close proximity to the dTSS, with S displaying the most constitutive and highest expression profile. Spiked hexamers occur in close proximity to the dTSS and low hexamers are enriched for those pyrimidine-rich hexamers found in Arabidopsis thaliana and Oryza sativa core promoters as the Y Patch. We found that E. grandis core promoters include those such as the TATA-box class which is conserved across kingdoms, the CT and GA classes, which are conserved in Arabidopsis, and a number of classes which, thus far, appear unique to Eucalyptus. We postulate possible underlying mechanisms of each core promoter class based on their sequence composition and suggest regulation by TBP binding (TA), nucleosome positioning (W), DNA stability (S), and non-BDNA conformation (CT and GA). This research provides a basal understanding of cistranscriptional regulation at the core promoter in this economically important woody plant species and provides insight into the mechanisms of permissive transcription across plant species.
Reverse vaccinology based identification of anti- Rhipicephalus microplus vaccine candidates
(University of Pretoria, 2013) Maritz-Olivier, Christine; Neitz, A.W.H. (Albert Walter Herman); christianstutzer@gmail.com; Stutzer, Christian
The rapid geographic expansion of the cattle tick species, Rhipicephalus microplus, as well as the introduction of diseases into previously unaffected areas and the increased incidence of acaricide resistance in this species are of great concern in Africa and Southern Africa. The feasibility of developing an effective program for tick control that includes vaccination of cattle with tick protective antigens has been demonstrated sufficiently with the Bm86 vaccine. To date, a number of candidates have been identified that have been tested in cattle vaccination trials, but none could infer greater protection compared to Bm86. Genomic approaches to study R. microplus has only recently became possible with the availability of EST and assembled sequences databases (i.e. BmGI database with 13, 643 unique transcripts). During this study, a custom-based oligonucleotide microarray chip was designed from available genomic data allowing the simultaneous transcriptional analysis of some 13,456 R. microplus transcripts. This tool allowed a global view of the transcriptome of various adult female tissues and the immature life stages of R. microplus during feeding on South African cattle breeds. Additional clustering and annotation of transcripts resulted in the identification of biochemical processes and protein families that are unique to or conserved for the different life stages. Consequently global gene expression indicated 85 transcripts as shared between all life stages, along with a number of transcripts that were life stage specific or shared between the life stages tested. A reverse vaccinology approach was followed and the transcriptome data was subjected to bioinformatic and immuno-informatic analysis. Consequently, a set of potential antigens were identified for future evaluation as anti-tick vaccines and the applicability of reverse vaccinology in rational anti-tick vaccine discovery was assessed.
Investigating the anti-inflammatory properties of epigallocatechin gallate from green tea Camellia sinensis (L) O.Kuntze in mammalian innate immunity
(University of Pretoria, 2020) Motshwene, Precious G.; Apostolides, Zeno; u17311706@tuks.co.za; Nyide, Nolwando Babalwa
Humans are hypersensitive to lipopolysaccharide, a gram-negative bacterial endotoxin that stimulates an inflammatory response by binding to the extracellular domain of TLR4 of innate immune cells. Through the MyD88-dependent pathway, NO (a signalling molecule) mediates the inflammatory response to clear pathogenic infection and promote wound healing. This pathway involves the activation of transcription factors, AP-1 and NF-κB, which promote the production of pro-inflammatory cytokines such as TNFα. However, dysregulated inflammation leads to chronic inflammation, causing autoimmune diseases including rheumatoid arthritis and asthma, among others. Considering the shortfalls of currently available treatments such as NSAIDs, there is a growing need for novel treatment strategies. Studies suggest that EGCG, a phenolic compound found abundantly in green tea (C. sinensis), possesses anti-inflammatory activity. This study sought to test this hypothesis. Firstly, 70% methanol was used to extract phenolic compounds and caffeine from three commercial green tea brands (Lipton, Tetley and BST) available on the South African market. Then the EGCG and caffeine contents of these green tea products were measured using UPLC analysis. Additionally, the total phenolic contents (TPCs) were measured for comparison using the Folin-Ciocalteu method. Secondly, the anti-inflammatory properties of EGCG were investigated in an acellular system and in RAW 264.7 cells using the Griess assay to test for NO production. Thereafter, MTT assays were conducted on the macrophages to determine cell viability after exposure to EGCG. Lastly, the role of EGCG on TLR4 signalling during inflammation was tested in the context of the MyD88-dependent pathway. To do this, NF-κB expression was measured using luciferase activity assays in LPS-stimulated HEK 293 cells treated with EGCG. Additionally, ELISAs were performed on stimulated wild-type immortalised bone marrow-derived macrophages (iBMDMs) to monitor the expression of TNFα in response to treatment with EGCG. This study determined the average total phenolic content of Lipton, Tetley and BST to be 18%, which falls within the range of 15 – 20% reported in previous literature. Lipton contained a significantly (P < 0.05) higher %TPC than each of the other brands. Furthermore, it was established that these brands contain between 0.8 – 0.9% caffeine and approximately 1% EGCG by dry weight. Although the literature indicated that the caffeine content was only slightly lower that the reported range of 1.1 – 2.0%, it was noted that the observed EGCG content was much lower than the expected value of 3%. Furthermore, EGCG exhibited the highest NO-scavenging activity at concentrations of 100 µM in an acellular system, and 200 µM in RAW 264.7 cells. Minimal cytotoxicity towards the cells was observed at a concentration of 200 µM, although previous literature showed that such a high concentration of EGCG was toxic to human astrocytoma U373MG cells. Moreover, TLR4 signalling assays revealed that 10 µM EGCG significantly down-regulated NF-κB expression in LPS-stimulated HEK 293 cells, however, EGCG had no significant effect on the expression levels of TNFα in LPS-stimulated iBMDMs. Hence, it was concluded that further study was required before a sound conclusion can be drawn regarding the role of EGCG in inflammation since the evidence was inconclusive.
Triazolyl Ru(II), Os(II), and Ir(III) complexes as potential HIV-1 entry inhibitors
(University of Pretoria, 2021) Gama, Ntombenhle; Meyer, Debra; bmfputterill@gmail.com; Putterill, Brandon Marquand Fraser
Background: The human immunodeficiency virus (HIV) is the cause of the acquired immunodeficiency syndrome (AIDS). AIDS is fatal if not treated appropriately. Although there are treatment options for the infection, there are many problems associated with it, including non-compliance to prescribed treatments due to toxicity and side effects, leading to drug resistance. There is therefore a need to develop novel drugs that are less toxic. This study contributes to the fight against HIV/AIDS by recommending new metallodrugs able to address the shortcomings of existing treatments. Metals have previously demonstrated potential in targeting HIV-1, mostly with activity against the enzymes reverse transcriptase and protease. The current study investigated the effects of metal-based complexes against viral entry into host cells. Methods: Three metal-based complexes; Aryl-1H-1,2,3- triazole-based cyclometalated Ruthenium (II) complex (A), Aryl-1H-1,2,3- triazole-based cyclometalated Iridium (III) complex (B) and Aryl-1H-1,2,3- triazole-based cyclometalated Osmium (II) complexes (C) were investigated for potential HIV entry inhibition and their activity was compared to that of the ligand which did not contain the metal component. The study analysed the toxicity of the complexes in TZM-bl cells and Peripheral blood mononuclear cells (PBMCs). Three pseudo-viruses (CAP 210, Du 156 and Q 23) were created using transformation and transfection methods and a luciferase reporter gene assay was used to analyse the inhibitory effects of the complexes on the pseudo-virus infection of TZM bl cells. Active complexes were further analysed for a potential mechanism of action through in silico docking. Results and discussion: The complexes were found to have lower CC50 values in PBMCs compared to TZM-bl cells. In both cell lines, B had the lowest CC50 value, which can be attributed to the increased hydrolysis of the chloride atom bound to the iridium as well as the increased uptake into the cells. Based on the luciferase reporter gene assay all three of the metal-based complexes had inhibition of viral infection with IC50 values ranging from 5.34 – 7.41 µM for A, 2.35 – 8.09 µM for B and 2.59 to 4.18 µM for C. The ligand was only analysed for any inhibitory activity on one of the pseudo-viruses (Du 156) and was found to have no significant inhibition. Selectivity index (SI) values indicated the complexes were effective at non-toxic concentrations with values ranging from 1.61 – 4.56 for B, 3.29 – 4.56 for A and 7.03 – 11.26 for C. In silico docking analysis of the proteins involved in viral entry indicated that inhibition possibly occurred through interaction with the CCR5 co-receptor, as the docking scores for this protein were the most negative indicative of favourable interactions between proteins and ligands. Conclusion: The metal-based complexes showed inhibition with IC50 values in the low micromolar range, while the ligand had no statistically significant inhibition. This suggests that the presence of the metal ion enhances viral inhibition. Furthermore, inhibition was through the interaction of the complexes with CCR5, in a manner similar to that of Maraviroc, a clinically utilized CCR5 inhibitor. This study identified novel metal-based HIV-1 entry inhibitors which were effective against HIV-1 subtype A and C at non-toxic concentrations.
In silico and in vitro inhibition of α- glucosidase and α-amylase by compounds from culinary herbs and spices, for possible treatment of type 2 diabetes
(University of Pretoria, 2020) Apostolides, Zeno; Bester, Megan J.; u15018131@tuks.co.za; Tolmie, Morné
Diabetes is one of the largest health challenges of the 21st century and is amongst the top 10 causes of death globally. There is no cure for type 2 diabetes (T2DM) and the search for new and improved treatments is ongoing. Various pharmaceuticals are available to treat T2DM, but with varied success. Many traditional herbal medicines are also used to treat T2DM, but mostly without scientific validation. Alternative treatment strategies, like herbal medicines and associated active compounds, can prove to be more cost-effective and may lack undesirable side-effects. This study aimed to assess the in vitro hypoglycaemic, toxicity and insulin mimicking effects of seven compounds found in commercially available herbs and spices, using in silico and in vitro relationship studies. These herbal compounds were acetyleugenol, apigenin, cinnamic acid, eriodictyol, myrcene, piperine and rosmarinic acid, they were chosen based on scientific reports on pleiotropic effects related to the inhibition of starch hydrolysing enzymes and insulin mimicking effects. Various in silico physiochemical properties of each compound was evaluated and compared with the antidiabetic drug, acarbose. All the herbal compounds had better drug-like features than acarbose. Candidate compounds were further analysed using the Search Tool for Interactions of Chemicals (STITCH) database to explore drug-target interactions, for possible harmful cross-reactions. The drug-target networks generated on STITCH showed no undesirable cross-reactions and highlighted the anti-carcinogenic and anti-inflammatory properties of the herbal compounds. The enzyme inhibitory nature was evaluated using in silico docking analysis with the Glide algorithm in the Maestro software and was further confirmed by in vitro α-amylase and α-glucosidase colorimetric assays. Alpha-amylase and α-glucosidase have been identified as important therapeutic targets for the management of T2DM. The inhibition of these enzymes would lead to a decrease in postprandial hyperglycaemia, however most clinically used drugs have undesirable side effects. Herbs and spices such as parsley, cinnamon, pepper, oregano, mint and cloves alleviate flatulence, diarrhoea and abdominal pain, counteracting the side effects commonly caused by α-amylase and α-glucosidase inhibitors. The in silico results identified which herbal compounds had better docking scores (more negative delta G values) than acarbose namely, apigenin, eriodictyol, piperine and rosmarinic acid. The in vitro studies revealed that all compounds, except myrcene, inhibited α-amylase and α-glucosidase in a dose-dependent manner. The Ki value of acarbose, a widely prescribed α-glucosidase inhibitor, and eriodictyol, apigenin and piperine were similar (p > 0.05). For α-amylase inhibition, the Ki value of acarbose and those of rosmarinic acid , apigenin and cinnamic acid were similar (p > 0.05). The relationship between the in silico and in vitro results correlated well, where a more negative docking score translated to a higher in vitro inhibitory activity. The effect of the herbal compounds on cell viability in C2C12 myotubes and HepG2 hepatocarcinoma cells, using the sulforhodamine B assay, was then determined. Eriodictyol and apigenin displayed noticeable toxicity against HepG2 and C2C12 cells. Acetyleugenol, cinnamic acid, myrcene, piperine and rosmarinic acid had similar (p > 0.05) IC50 values to acarbose in both cell lines. Fluorescence detection was used to investigate the effects of each herbal compound on glucose uptake in HepG2 and C2C12 cells. All of the compounds significantly increased glucose uptake in these cell lines, compared to the control (p < 0.05), with efficacy in the same order as the positive control, insulin (p > 0.05). This study provides evidence for the antidiabetic potential of herbal compounds in terms of their ability to prevent post-prandial hyperglycaemia, through the inhibition of starch hydrolysing enzymes, and alleviate hyperglycaemia by mimicking the action of insulin. The most promising compounds were cinnamic acid, piperine and rosmarinic acid. Using herbs and spices would have several advantages, including their widespread availability, easily cultivatable nature, affordability and health benefits. These compounds can easily be consumed through teas or using herbs and spices to flavour food.
Metabolomic markers for agronomic traits and their possible biochemical mechanisms in black tea Camellia sinensis (L.) O. Kuntze
(University of Pretoria, 2020-11-17) Apostolides, Zeno; cnyarukowa@gmail.com; Nyarukowa, Christopher
Climate change is causing droughts, which are affecting crop production globally, and disrupting plant metabolism. Due to the unpredictable natural droughts that occur, causing tea farmers significant losses in tea estates, a Short-time Withering Assessment of Probability for Drought Tolerance (SWAPDT) method for distinguishing between drought tolerant (DT) and drought susceptible (DS) Camellia sinensis cultivars was developed based on cultivars from the Tea Research Foundation for Central Africa in Malawi, and validated on 400 samples from the Tea Research Institute in Kenya. From the results, a sample size of 20 tea trees was deemed sufficient to accurately determine the drought susceptibility of a large tea field of approximately 5 - 20 hectares, containing 50 000 - 200 000 tea trees, were the difference between their mean values is approximately 6%. Tea production and subsequently its quality rely on evenly distributed rainfall. Tea consumers concern themselves with the quality of tea, in particular its flavour and aroma. To breed for these phenotypic traits is challenging due to these being qualitative traits inherited from parents, and influenced by environment. Two C. sinensis populations, 60 Commercial cultivars and 250 NonCommercial cultivars (TRFK St. 504 and TRFK St. 524) were employed in a part of this study to identify the Quantitative Trait Loci (QTL) responsible for yield, drought tolerance and quality centred on a genetic map constructed using the DArTseq platform. The map comprised 15 linkage groups analogous to chromosome haploid number of tea plant (2n = 2x = 30) and spanned 1260.1 cM with a mean interval of 1.1 cM between markers. Sixteen phenotypic traits were evaluated in both populations, and three, 11 and 46 putative QTLs were discovered after mapping on the 15 linkage groups, associated with tea quality from Gas Chromatography-Mass Spectrometry (GC-MS), Nuclear Magnetic Resonance (1H-NMR) and Ultra-Performance Liquid Chromatography (UPLC) data respectively. The variance explained by the QTLs varied from 4.6 to 96.3%, with an average of 28%. Using the KEGG database, the putative QTLs linked to yield, drought tolerance and quality were secondary metabolites associated with tea phenolic biomolecules and abiotic stress. Principal Component Analysis was performed on the GC-MS, 1H-NMR and UPLC data, and from these, the UPLC data showed clearer separation and clustering between the Commercial and NonCommercial cultivars. With focus on the UPLC data, it was narrowed down to the five catechins, four theaflavins and caffeine; these were used to develop several logistic regression models. The model based on only the fresh leaf catechins classified over 90% of the 310 genotypes as either Commercial or NonCommercial cultivars. This model may be useful in predicting the suitability for commercialization of promising selections from mature seedling fields, based on the analysis of their dried green leaves. Last, 20 Commercial and 20 NonCommercial cultivars were analysed using UPLC-MS. New metabolites were identified as contributing to drought tolerance, yield and higher quality of the Commercial as compared to the NonCommercial cultivars.
The contribution of Plasmodium falciparum genetic diversity to differentiate drug response in gametocytes
(University of Pretoria, 2020) Niemand, Jandeli; Birkholtz, Lyn-Marie; u13243099@tuks.co.za; Maboane, Suzan
Late-stage gametocytes of P. falciparum parasites are responsible for the ongoing human-to-mosquito transmission of malaria disease. To block transmission, novel gametocytocidal compounds are required. It is thus important to determine ex vivo efficacy against diverse, contemporary clinical isolates as an early filter to provide confirmation of novel gametocytocidal activity in the field. This study hypothesizes that differences in drug responses in late-stage gametocytes of clinical isolates can be correlated with the extent of genetic diversity of clinical isolates. Previously, potent phosphatidylinositol 4-kinase (PI4K) inhibitors indicated differential drug response, which was observed in transmissible stages of ex vivo southern African P. falciparum clinical isolates. These differential drug responses were not only limited to kinase inhibitors, but also seen with endoperoxide and ATP4 inhibitors that were used in the current study. Microsatellite (MS) and single nucleotide polymorphism (SNP) markers determined the allelic variation of clinical isolates, and therefore genetic complexity can clarify differential drug response in isolates. Here we confirm that isolates from the high transmission areas are characterised by a high multiplicity of infection (MOI) and isolates exhibit many unique alleles. This study also shows that there is a relationship between gametocyte production and isolates that are genetically diverse. Furthermore, the study presents SNP barcoding as a more sensitive and robust genotyping technique, as it was able to correlate differential drug response to genetic complexity of clinical isolates.
Novel acid-labile and targeted nanoparticles as possible antimalarial drug delivery systems
(University of Pretoria, 2020) Birkholtz, Lyn-Marie; Coertzen, Dina; u13062922@tuks.co.za; Leshabane, Meta Kgaogelo
The multistage life cycle of malaria-causing P. falciparum is complex, making prevention and treatment difficult. As a result of resistance to many antimalarial drugs, novel compounds with unexplored targets are constantly sought after for the purpose of treating the symptoms of malaria. Here, novel compounds were screened for antiplasmodial activity against the symptom-causing asexual intraerythrocytic malaria-causing parasites. Unfortunately, many novel compounds in the drug discovery pipeline and drugs in clinical use possess underlying pharmacological issues that makes administration challenging. These include low aqueous solubility and short half-life which negatively impact bioavailability resulting in toxicity. This, in turn, increases patient non-compliance and the emergence of drug-resistant strains. Nanoparticles (NP) have the ability to mask drugs from the external environment while increasing circulation time and often alleviate many issues at once. Furthermore, the selected drugs do not need to be modified. Drug conjugation NPs with a targeting ligand and stimuli-responsive linkers have been extensively researched in many diseases, however, none have been reported for malaria clinically. Here, the first acid-labile targeted NP (tNP) that exploits the biology of infected erythrocytes and the specialised food vacuole (FV) of P. falciparum is interrogated for ability to decrease toxicity while retaining antimalarial activity. This dissertation describes the effect of tNPs on the efficacy and toxicity of selected compounds. In vitro haemolysis and cytotoxicity assays revealed that the tNPs are biocompatible to erythrocytes and HepG2 cells. The data also shows that tNPs decrease the toxicity of drugs and the chosen novel compound against human cells. A decrease in antiplasmodial activity was observed in vitro for the tNPs when compared to the novel compound and drugs on their own. However, this was due to the biogenesis of the FV and a shortened window of release. Nonetheless, the NP backbone was not active against P. falciparum intraerythrocytic parasites whereas tNPs were, showing activity due to released drug. The targeting ligand was also not specific for antiplasmodial activity. Although a significant loss in activity is observed, the results presented here suggests that these novel acid-labile tNPs serve as an attractive starting point for targeted treatment of malaria with an improved patient tolerance. Furthermore, novel compounds with issues can be selected without having to be modified or completely discarded. Therefore, increasing the chances of finding a variety of compounds that can be used to treat malaria while keeping patients safe.
Metabolomic analysis of maize (Zea mays L.) seedlings treated with selected plant growth promoting rhizobacteria
(University of Pretoria, 2019) Apostolides, Zeno; Labuschagne, Nico; u13039042@tuks.co.za; Otukpa, Faith Obiye
Global food production has significantly increased mainly due to the use of high-yield crop varieties, fertilisers, fungicides and pesticides, and improved irrigation methods. Despite the increase in production, there has been a significant cost to the environment in the form of pollution, and to farmers in the form of rising fertiliser and pesticide costs. The impacts on the environment include but are not limited to groundwater contamination, declining soil health and increased pest and pathogen resistance, all which increase the financial cost to farmers. To reverse or rather salvage the situation, more sustainable agricultural practices need to be employed that will maintain high productivity with little to no damage to the environment, and will reduce agrochemical use, thus, reducing the financial strain on farmers. A potential solution would be to exploit soil dwelling rhizospheric microorganisms to improve plant growth with little to no application of agrochemicals. The rhizosphere refers to the region of soil directly influenced by plant roots and is home to microorganisms known as plant growth promoting rhizobacteria (PGPR). These PGPR have been found to stimulate plant growth via numerous mechanisms, which directly affect the plant metabolome and in turn translates into observable effects in the plant phenotype. The plant metabolome has been described as the bridge between the genotype and phenotype, thus metabolomics acts as a useful tool to evaluate the contribution of external influences on the plant phenotype based on metabolic changes. The effect of PGPR on the plant metabolome is vital in understanding their mode of action, which will further validate their use in farming. The overall aim of this project was to assess the effect of selected PGPR strains with known plant growth promoting activity on the metabolic profile of maize seedlings; and to evaluate if these changes in the metabolic profile directly correlate with the observable effects on the growth of the seedlings. To achieve this aim, firstly, the effect of Lysinibacillus sphaericus (T19), Paenibacillus sp. (T29) and Bacillus megaterium (A07) on early maize growth, i.e., the effects on dry root and shoot biomass, leaf chlorophyll content, stem diameter and shoot length, was assessed. Secondly, the effect of single strain PGPR inoculation on the metabolic profile of maize was evaluated. Finally, metabolomics analysis was conducted on the roots and shoots of the maize seedlings inoculated with strains T29 and T19 respectively. Statistical analysis of the metabolomics results was conducted to find significant pathways and discriminating metabolites between the control and inoculated plants. To assess the effects of the PGPR strains on early maize growth, a greenhouse trial was first conducted. Non-invasive techniques were used to measure the growth parameters that could be recorded prior to harvest. Next an untargeted metabolomics approach was used to analyse the metabolome of harvested roots and shoots. Metabolomics data acquisition was achieved using ultra-performance liquid chromatography hyphenated to quadrupole time of flight mass spectrometry detection (UPLC/QTOF-MS). Finally, to evaluate the effect of single strain inoculation on the maize root and shoot metabolome, univariate and multivariate methods were applied. The results of the greenhouse trial showed a tendency of strains T19 and T29 in stimulating shoot growth and root growth respectively in the maize seedlings. Pathway analysis using results from univariate analysis revealed a number of pathways affected by T19 and T29 in the shoots and roots respectively. Multivariate statistical analysis also showed that the inoculated samples differed from the control samples, albeit with varying trends, which indicates differing metabolic states. Some of the metabolic pathways deemed significant in the inoculated shoots and roots were amino acid, nucleotide metabolism or carbon fixation related. A number of discriminating features were found to be differentially regulated in the inoculated roots and shoots. Overall, the results showed that T19 and T29 inoculation stimulated metabolic responses in maize shoots and roots linked to plant growth and development.
Exploring the evolution of drug resistance in mycobacterium using whole genome sequencing data
(University of Pretoria, 2019) Reva, Oleg N.; u17288632@tuks.co.za; Muzondiwa, Dillon
Mycobacterium tuberculosis (Mtb) remains a global challenge that has been worsened by the emergence of drug resistant strains of Mtb. We used publicly available Next Generation Sequencing (NGS) and drug susceptibility (DST) data to develop “Resistance sniffer”, an online software program for the rapid prediction of lineage and Mtb drug resistance. Based on the distribution of polymorphisms in the genomes of Mtb, we calculated the power of association between the polymorphisms in different clades of Mtb and resistance to 13 anti-TB drugs. Our data suggests that the development of drug resistance in Mtb is a stepwise process that involves the accumulation of polymorphisms in the Mtb genome. We carefully curated the polymorphisms based on their association powers to create a diagnostic key that captures patterns of these polymorphisms that can be used to predict lineage and drug resistance in Mtb. This diagnosis key was incorporated into the Resistance Sniffer tool, an online software program that we developed for the rapid diagnosis of drug resistance in Mtb. The tool was tested using sequence data from the South Africa Medical Research Council (SA-MRC). Our data suggests that the majority of the strains in SA may have been brought by the arrival of European settlers while the more resistant strains may have been introduced in the region by Asian travellers later on. We next sought to determine non-random associations between polymorphic sites in genomes of Mtb. Using the attributable risk (Ra) statistical methods, we distinguished between functional associations and associations that may have been due to genetic drift events for different Mtb clades. We then integrated the (Ra) data with drug susceptibility and annotation data to generate networks in Cytoscape 3.71. These networks were then used to infer evolutionary trajectories that drive the emergence and fixation of the drug resistant phenotype in different clades of Mtb. We demonstrate that strains from the Lineage 1.2 are associated with less complex functional associations compared to the strains from other clades such as the Asian and Euro-American clades. Our data also shows that the predisposition of strains from the Asian clades to develop multi-drug resistance may be attributed to a complex network of functional interactions of mutations in genes that are involved in several aspects of Mtb physiology such as cell wall modelling, lipid metabolism, stress response and DNA repair.
Metabolic profiling for identification of potential biomarkers in potato tuber extracts associated with tolerance to Spongospora subterranea f. sp. subterranea infection
(University of Pretoria, 2019) Apostolides, Zeno; Van der Waals, Jacquie E. (Jacqueline Elise); u15056237@tuks.co.za; Modisane, Gontse Kehumile Joyce
Potatoes are popular and reliable crops all over the world. Unfortunately, its production is threatened by detrimental factors such as fungi, bacteria and viruses that attack potatoes during growth and storage. Among all the factors infecting the potato crops, the fungal disease is the most important diseases that cause severe damage in potatoes. Spongospora subterranea f. sp. subterranea (Sss) is one of the fungal pathogens of economic importance in potato production, causing a significant reduction in yield and quality of potato tubers globally. Currently, there are no effective control methods available for this pathogen, partly due to the longevity of the highly resistant sporosori surviving in the soil. The use of cultivars resistant to Sss is the most efficient and long-term strategy for preventing the build-up of field inoculum and the spread of the pathogen. However, classical breeding and selection methods are laborious, costly and time-consuming processes. Metabolomics, as a new advanced technology, was used to better understand the biochemical mechanisms of tolerance and to identify metabolic biomarkers that could distinguish between tolerant and susceptible cultivars with statistical significance at p≤ 0.05. Four susceptible and six tolerant cultivars of five biological repeats were each grown in the greenhouse inoculated with Sss pathogen-suspension and mock-solution. Based on the results, multivariate data analysis revealed different responses to Sss infection when comparing tolerant with susceptible cultivars. Most of the metabolites identified in the Sss-inoculated samples were abundant in tolerant compared to susceptible cultivars such as amino acids, phenylpropanoids, fatty acids, organic acids and alkaloids. These metabolites are known for their antimicrobial properties as well as cell wall thickening, used to suppress pathogen development in plants, leading to reduced susceptibility. In conclusion, palmitic acid, 3-indole acrylic acid and cuscohygrine were found as possible biomarkers for tolerance against Sss infection in potato tuber extracts. These biomarkers may play an important role in potato tolerance to Sss.

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