Theses and Dissertations (Biochemistry, Genetics and Microbiology (BGM))

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Mitochondrial genomes from the Ceratocystidaceae and species boundaries in Ceratocystis
(University of Pretoria, 2024-11) Duong, Tuan A.; Wingfield, Brenda D.; Kanzi, Aquillah M.; anien95@gmail.com; Viljoen, Anien
The fungal family Ceratocystidaeceae contains several genera that are considered highly pathogenic and able to cause disease on a wide range of hosts. The taxonomic history of Ceratocystidaceae species has been under debate for many years due to the lack of morphologically distinguishing characteristics between the species and genera. Chapter one of this thesis provides a broad overview of the speciation process from defining species to studying the barriers that prevents gene flow between populations or prevents hybrid zygotes from developing before and/or after fertilization. Different speciation genomic approaches, their advantages and limitations are also discussed. The chapter also provides a brief overview of the taxonomy, species delineation and the controversies associated with this process in the Ceratocystidaceae, focusing specifically on the Ceratocystis fimbriata sensu lato complex. The second chapter presents the work on characterisation of mitochondrial (mt) genomes for 18 species in 10 genera in the Ceratocystidaceae. The aim of this chapter was thus to assemble, characterise and comparatively analyse mt genomes from multiple species and genera in the Ceratocystidaceae. The work of chapter 3 focuses on the taxonomic confusion that has historically plagued the C. fimbriata s.l complex. The aim of this chapter was to use whole genome SNP data from a large collection of isolates of C. fimbriata sensu lato complex to investigate species boundaries, phylogenetic relationships, and the genetic differentiation between these closely related species. Taken together, the body of the work presented in this dissertation contributes to the current understanding of mitochondrial genome evolution, species boundaries, and the evolutionary relationship among species and genera within the Ceratocystidaceae. These findings pave the way for future research aimed at exploring the biology, pathogenicity, speciation, and host adaptation of the important group of fungi.
Modelling the population dynamics of CRISPR-Cas9 gene drive systems in Sirex noctilio
(University of Pretoria, 2024-11) Slippers, Bernard; Chapwanya, Michael; Ouifiki, Rachid; hannes.strydom1995@gmail.com; Strydom, Hannes
Sirex noctilio is an invasive pest of pine that has caused significant economic damage in South Africa and many other Southern Hemisphere countries. Current management tools are not efficient in all cases and consequently there is a need for more efficient and targeted control measures. An emerging tool for pest management is the use of gene editing and associated gene drive systems. In this study, we aim to investigate the use of CRISPR-Cas gene drive systems in the management of S. noctilio in South Africa. As a first step, we developed a model for the population dynamics of S. noctilio, using historical national population monitoring data and incorporating the influence of two main biological control agents of the pest. We then modelled the influence of two different CRISPR-Cas systems on the population dynamics of S. noctilio namely, a baseline CRISPR model and Complementary Sex Determination CRISPR (CSD) model. Each model is used to simulate a male and female only introduction strategy to estimate the effectiveness of different methods of introducing the gene drive system. Results suggest that both CRISPR gene drive system would be effective at controlling the population growth of S. noctilio at high levels of introduction, but overall population control would be hindered by practical limitations. Although only two CRISPR models were explored, the underlying population model serves as a framework for further studies into the population dynamics of Sirex noctilio, as well as many other CRISPR-Cas gene drive systems.
In vitro validation of chemo-transcriptomic fingerprints for the classification of the mode of action of antimalarial drugs
(University of Pretoria, 2024-10) Birkholtz, Lyn-Marie; u19010126@tuks.co.za; Venter, Natanya
Malaria is an infectious disease brought on by Plasmodium parasites. Resistance development to current treatment measures is a significant challenge to the progress made in eradicating malaria. As a result, developing new drugs with novel targets and modes of action (MoA) is of utmost importance to address this resistance. van Heerden et al. used classification-based machine learning to develop a rationally selected model capable of stratifying compounds into different MoA groups with a 77 % accuracy. This model used chemo-transcriptomic fingerprints to indicate that the variant expression of only 50 transcripts was sufficient to classify different compounds with similar MoA into the same subsets quickly and specifically. This study used real-time, quantitative PCR and the 2-△△Cq relative quantification method to investigate the in vitro expression levels of the biomarkers that van Heerden et al. identified as responsive to compound treatment within parasite populations treated with antimalarial compounds. Five control compounds, each with a known MoA was used to established that the qPCR amplification of the biomarkers was sufficient to distinguish between the compounds and that compounds with specific targets clustered separately from those with non-specific targets. Four clinical candidates with dissimilar MoAs not previously evaluated in the machine learning model solidified that the biomarkers could create distinctive chemo-transcriptomic fingerprints for each compound’s MoA. Lastly, a clinical candidate that shares a target with a control compound was introduced, proving that compounds with overlapping biological activity showed similarities in their chemo-transcriptomic fingerprints. This data indicated that the biomarkers identified using machine learning could be predictive biomarkers for compound MoA classification. The limited number of biomarkers, as well as the established qPCR-based platform parameters in this study, provides a rapid and scalable means to determine a compound’s MoA, therefore greatly benefiting antimalarial drug discovery by allowing drug candidates to be evaluated for unfavourable MoAs and ensuring that the MoA remains unchanged during lead optimisation.
The effect of mpc1/mpc2 overexpression in intraerythrocytic Plasmodium falciparum parasites
(University of Pretoria, 2024-11) Niemand, Jandeli; Birkholtz, Lyn-Marie; u18001964@tuks.co.za; Voges, Suzelle
Pyruvate functions as a metabolic switch between aerobic and anaerobic metabolism, allowing a shift to an alternate metabolic pathway when required. The mitochondrial pyruvate carrier heterocomplex (MPC), composed of MPC1 and MPC2, has been identified as the transport complex responsible for pyruvate transport into the mitochondria. In P. falciparum, the putatively annotated mpc1 (pf3d7_1340800) and mpc2 (pf3d7_1470400) genes have yet to be characterised. In this study, the mpc1 and mpc2 genes in P. falciparum parasites were investigated using an overexpression approach. A transgenic P. falciparum parasite line constitutively expressing mpc1/mpc2 above basal level was established, and increased MPC abundance was confirmed. The transgenic parasites were then compared to the wild-type to confirm that the genetic modification allowing the mpc1/mpc2 overexpression did not negatively affect intraerythrocytic parasite proliferation, survival, or morphology. Likewise, mitochondrial viability, abundance of other mitochondrial metabolism proteins, and parasite sensitivity to compounds inhibiting mitochondrial function was also unaffected. To gain insight into the biology surrounding the activity of the mpc1/mpc2 genes, the downstream effects of mpc1/mpc2 overexpression in the transgenic and wild-type P. falciparum parasites lines were compared. Chemical interrogation with an MPC inhibitor indicated a reduction in parasite sensitivity to the inhibitor upon mpc1/mpc2 overexpression, whereas no difference in the parasite sensitivity was observed when treated with other types of inhibitors. The overexpression of mpc1/mpc2 resulted in reduced lactate production, as expected, since less pyruvate would remain in the cytosol to be converted into lactate. Additionally, mpc1/mpc2 overexpression promoted parasite survival during glutamine starvation as expected, since more pyruvate can enter the mitochondria to drive the TCA cycle to compensate for the lack of glutamine. Therefore, increased mpc1/mpc2 expression resulted in biological changes consistent with the expected biological responses of increased MPC activity, confirming the annotation of these genes as a MPC in intraerythrocytic P. falciparum parasites.
Genomics insights into the global evolution and antibiotic resistance of the Mycobacterium tuberculosis complex
(University of Pretoria, 2025-02) Reva, Oleg; u17288632@tuks.co.za; Muzondiwa, Dillon
Mycobacterium tuberculosis (Mtb) recently reclaimed its status as the leading global cause of death from a single infectious agent after three years of COVID-19 holding the top position. We used Mtb whole genome sequencing data (WGS) to explore the diversity of human-adapted lineages of Mtb. Using publicly available datasets, we curated and characterised a large global WGS dataset of more than 9000 Mtb strains sampled across the globe. Based on the distribution of single nucleotide polymorphisms, we performed lineage classification, drug resistance predictions and molecular clock estimations to characterise the global evolution of Mtb and create a non-redundant global reference dataset. Our data suggested that public Mtb WGS datasets are highly redundant, and redundancy minimisation is required before analysing large datasets. We next sought to explore the evolutionary dynamics that shaped the genetic landscape of the African continent which has been suggested as the origins of Mtb. We demonstrate that Lineage 2 and Lineage 4 are the most dominant on the continent. Using Maximum Likelihood and Bayesian phylogenetic techniques, we mapped identified drug resistance-associated mutations to time-resolved phylogenies. We estimated that drug resistance on the continent emerged at multiple events, with the earliest emergence of drug resistance occurring in the mid-20th century. We also identified the presence of resistance mutations associated with recently introduced drugs in isolates that were sampled prior to the use of these drugs. Using Bayesian skyline coalescent inference, we observed an expansion in the Mtb population in Africa in timelines that coincided with increased migration from Europe and Asia into Africa. We also inferred a population expansion of Mtb at the time when HIV prevalence was at its peak on the continent. We next sought to understand the evolutionary dynamics of Lineage 2 and Lineage 4 Mtb in the Southern Africa Development Community (SADC) region, a part of the continent which carries the highest burden of HIV/TB coinfection. We demonstrate that the heterogeneity of Mtb Lineage 2 diversity in the SADC region is under-characterised. We identify 13 sublineages of Lineage 2 in the region from our analysis. To explore the origins of SADC Lineage 2 and Lineage 4, we employed two phylogeographic approaches and both of them place East Asia as the origin of Lineage 2 and Europe as the origin of Lineage 4. We also infer that the two lineages were introduced through multiple introduction events with South Africa as a central hub for the dispersion of the lineages northwards. Taken together, our phylogeographic analysis and our Bayesian skyline results suggest that migration and colonialism played a role in shaping the diversity of the two Lineages in SADC. Lastly, using mathematical models, drug susceptibility testing data and genomic data, we sought to model the epistatic dynamics that govern drug resistance in Mtb. We obtained co-dependency estimates that represent the probability of one mutation emerging after another mutation. We then created networks and traced the trajectories from drug susceptibility status to pre-XDR-TB status.
Repositioning the antitubercular candidate SQ109 as potent antimalarial with polypharmacology
(University of Pretoria, 2024) Birkholtz, Lyn-Marie; savjadewatson@gmail.com; Watson, Savannah Jade
Elimination of malaria requires effective targeting of the asexual replicative (asexual blood stage, ABS) and transmissible stages of the most lethal human malaria parasite, Plasmodium falciparum. Targeting gametocytes for transmission-blocking activity is particularly desirable as they are amenable to pharmacological intervention [1]. The search for new transmission-blocking antimalarial compounds has generally been biased towards compounds targeting biology important to the ABS parasites with screening campaigns prioritising hits based on ABS activity and gametocyte activity merely adding dual activity value to these drugs [2]. This approach poorly identifies equipotent or gametocyte-targeted antimalarial candidates for formulation into combination therapies for dual-stage activities. Alternative strategies used de novo, parallel screening against multiple life cycle stages, to identify the antitubercular clinical candidate MMV687273 (SQ109) with preferential activity against P. falciparum late-stage gametocytes [3] with comparatively poor activity against ABS parasites. Although combining compounds with gametocyte and ABS activity reduces the risk of resistance transmission, the pursuit of compounds solely focused on blocking transmission cannot currently be prioritised over the discovery of compounds with additional ABS activity given the urgent need for effective clinical treatments for malaria. Therefore, identifying compounds with dual-stage activity remains equally important. SQ109, therefore provides a new chemical starting point for further development and optimisation for dual-stage active compounds. Therefore, this study aimed to design and synthesise analogues of SQ109 to identify features required to consolidate curative and transmission-blocking compounds in structure activity relationship analysis, and to see if improvements can be made to ABS activity. Here, we confirmed SQ109’s antiplasmodial activity and evaluated the structure activity relationship of the SQ109 scaffold to design dual-stage active antiplasmodial compounds. We show that equipotent activity (IC50) in the 100 – 300 nM range could be attained for both ABS parasites and sexual stages, with the activity of most compounds retained against a multidrug-resistant strain. The dual-stage activity profile relies on high lipophilicity due to the adamantane headgroup, and antiplasmodial activity is critically dependent on the diamine linker. We additionally validated that this series could block transmission to mosquitoes, marking these compounds as novel chemotypes with dual-stage, antiplasmodial activity. These results indicate that this series holds promise as an effective treatment, with no apparent chemical features that could impede its development into a drug. SQ109 is a second-generation ethylenediamine antitubercular that completed phase IIb clinical trials for tuberculosis, and potently targets multidrug-resistant Mycobacterium tuberculosis [1, 4]. Its microbial target was identified as mycobacterial membrane protein large 3 [5], involved in cell wall biosynthesis. Additionally, SQ109 shows polypharmacology, whereby it triggers multiple biological responses and/or has multiple targets [6, 7]; as an example it was implicated in the disruption of the proton motive force [8] by acting as a protonophore uncoupler in mycobacteria. This activity has been proposed to be a significant driver of the mode of action of SQ109 against organisms that lack a mycobacterial membrane protein large 3 counterpart, such as Trypanosoma cruzi and Leishmania spp [9, 10], where effects on Ca2+, H+ and sterol homeostasis have also been found. No mycobacterial membrane protein large 3 homologues are present within the genome of P. falciparum parasites, and the Niemann–Pick type C1-related H+/lipid symporter, which belongs within the same resistance-nodulation-division superfamily of transporters as mycobacterial membrane protein large 3, was excluded as a potential mycobacterial membrane protein large 3-like target [3]. Therefore, with the mode of action of SQ109 in P. falciparum parasites essentially unknown, guided by its activity in other organisms, this study aimed to determine the mode of action of SQ109 and investigate if polypharmacology of SQ109 exists in P. falciparum parasites. If the polypharmacology of SQ109 held true for P. falciparum parasites, such a compound when used in combination, would have the added advantage of protecting compounds with ABS activity from resistance spreading events. Here, we proved polypharmacology of SQ109 in P. falciparum parasites, where its effects are based on physical properties rather than direct protein inhibition. These attributes of SQ109 are highly favourable for the development of more resistance-resistant antimalarials.
Gene structure and diversity analysis of the cattle tick vaccine antigen, TC-X
(University of Pretoria, 2024-11) Maritz-Olivier, Christine; Stutzer, Christian; u19026031@tuks.co.za; Tadmor, Cale
The cattle tick species Rhipicephalus microplus represents a major ectoparasite within the cattle industry, characterized by its rapid global dispersion, driven by its short life cycle, high reproductive capacity, and ability to adapt to diverse climatic conditions. This species is of considerable global significance due to the substantial economic losses it incurs through increased infestations and the transmission of tick-borne diseases (TBDs), which are associated with elevated morbidity and mortality rates in cattle. Chemical acaricides remain the primary method for tick control; however, their overuse has resulted in resistance across multiple tick species, prompting the exploration of alternative control strategies, such as vaccination. Currently, only one antigen, Bm86, has been commercialized for tick vaccination. Nevertheless, the efficacy of Bm86-based vaccines is geographically variable and necessitates multiple booster immunizations per season, underscoring the need to identify additional protective antigens and develop vaccines that provide long-term immunity. Our research has identified TC-X, a Kunitz protein from R. microplus, as a potential protective antigen in cattle vaccine trials. The combination of TC-X with a second antigen (Antigen 1) has been shown to induce immune memory and elicit a tick-mediated enhancement of the immune response in cattle. However, the gene structure, phylogeny, function, and diversity of TC-X remain poorly understood. In this study, we delineate the gene structure of TC-X, propose its potential roles in tick development and feeding, and demonstrate that the epitope regions predicted from TC-X exhibit low diversity, particularly within Kunitz domain 1. Furthermore, we suggest that TC-X may offer cross-protective benefits against other Rhipicephalus species, as it shares substantial protein identity with homologous proteins in these species. Future research will focus on expanding our RNA sequencing database to explore the global diversity of TC-X further. Ultimately, integrating vaccination strategies with acaricide treatment may reduce tick burdens, mitigate tick-borne diseases, and lessen environmental impacts, providing significant economic benefits to livestock producers.
Genetically diverse Plasmodium falciparum Southern Africa isolates provides insights into antimalarial drug sensitivity and gametocyte biology
(University of Pretoria, 2024-10) Birkholtz, Lyn-Marie; Van der Watt, Mariëtte; u14006325@tuks.co.za; Greyling, Nicola
Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum. Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.
Exploring the role of callose depositions in the resistance of Persea americana against root rot
(University of Pretoria, 2024-11) Swart, Velushka; Van den Berg, Noelani; moodleyshalya@gmail.com; Moodley, Shalya
Phytophthora root rot caused by Phytophthora cinnamomi threatens sustainable avocado production in South Africa. One of the most effective control methods for this disease is the use of partially resistant avocado rootstocks. To date, no fully resistant rootstocks have been developed, so the selection process for rootstocks that are more resilient against root rot diseases remains ongoing. It takes approximately 15-20 years for a new rootstock to be commercially released thus, to improve selection efficiency, it is crucial to identify key characteristics of partially resistant rootstocks that can be exploited to allow for a more targeted selection approach. Callose is a β-1,3 glucan deposited in cell walls, to create a physical barrier preventing pathogen invasion into the cell. Previous studies provide qualitative evidence, through confocal microscopy, that callose deposition may be a hallmark of partial resistance in response to P. cinnamomi infection. The aim of this study was to optimise a fluorometric assay to quantify callose production in avocado roots and then utilise this assay to determine if callose production is induced in response to P. cinnamomi infection. The study also sought to determine if callose production is a key characteristic of avocado rootstocks that are partially resistant to P. cinnamomi. This was achieved through chemical inhibition using 2-deoxy-D-glucose (2-DDG) prior to P. cinnamomi inoculation and assessing the resulting disease severity. This project comprised of two research chapters working towards elucidating the role of callose in avocado defence against P. cinnamomi. In the first chapter, the conditions for 2-DDG treatment were optimised, as this was the first study to use this inhibitor on avocado roots. The main objectives were to determine the optimum concentration and duration of 2-DDG treatment required to 1) successfully inhibit defence-related callose production and 2) ensure that 2-DDG did not negatively affect plant health. A secondary objective of this chapter was to optimise a callose extraction protocol from avocado roots to allow for fluorometric callose quantification with minimum background interference. The protocols developed were utilised in a subsequent research chapter, which tested the effect of callose inhibition on avocado rootstocks of varying tolerance levels, namely, one susceptible (R0.12) and two partially resistant (Dusa® and LeolaTM) rootstocks. The objectives of this chapter were to 1) evaluate if callose is produced in partially resistant avocado rootstocks in response to P. cinnamomi, and 2) determine the effect of callose inhibition in these rootstocks. The study yielded evidence for the induction of callose production in avocado roots in response to P. cinnamomi infection. Another significant contribution of the study was the development of a 2-DDG treatment regimen optimised for callose inhibition in avocado roots. Surprisingly, no differences in callose production were observed among different rootstocks with varying levels of tolerance to P. cinnamomi between 1) inoculated and uninoculated plants and 2) 2-DDG treated and untreated plants. The detection of a second avocado root pathogen, Phytopythium vexans, in the roots of the trial plants contributed to these confounding results. Pp. vexans infection was confirmed morphologically and molecularly using a TaqMan qPCR probe assay optimised for avocado roots. The impact of Pp. vexans on disease severity and callose measurements were subsequently assessed. While some results from this study were inconclusive, the findings highlighted the complexity of avocado defence, suggesting that callose production could be a general defence pathway that is activated in response to infection by root rot pathogens. It may become increasingly important to understand the interplay between various pathogens within a root rot complex rather than infection by P. cinnamomi alone. Future research should consider host-pathogen interactions in the context of the avocado microbiome to provide a holistic understanding of the different factors that contribute to plant defence in the field.
Detection, dispersal and global distribution of Ceratocystis species on Eucalyptus
(University of Pretoria, 2024-10) Barnes, Irene; Wingfield, Michael J.; u14049423@tuks.co.za; Lynn, Kira Mary Theresa
In this thesis, Detection, dispersal, and global distribution of Ceratocystis species on Eucalyptus, the candidate investigated the species boundaries and transmission mechanisms of two economically important fungal pathogens. Utilizing a dataset of 1174 isolates collected from 11 countries and 8 plant hosts, population genetics, phylogenetic analysis, and morphological comparisons were employed to confirm that Ceratocystis manginecans and C. eucalypticola are distinct species, resolving long-standing taxonomic confusion. Hybridization events between these species were also identified clarifying ambiguity in the literature. The candidate developed a cutting-edge qPCR-based HRMA diagnostic tool enabling rapid species identification of these fungal pathogens directly from infected plant material and fungal cultures. This tool was successfully applied to reveal key dispersal mechanisms, including insect-mediated and airborne transmission. These findings offer valuable insights into pathogen detection, dispersal, and species boundaries, providing crucial tools for effectively managing the spread of these economically significant pathogens within the global forestry industry.
The efficacy of mycolic acid-enhanced PLGA nanoparticles for rifampicin delivery in tuberculosis treatment
(University of Pretoria, 2024-08) Lemmer, Yolandy; Verschoor, Jan Adrianus; kruger.goosen@gmail.com; Goosen, Kruger
This study explored the efficacy of nanoparticle-encapsulated rifampicin formulations for treating high Mycobacterium tuberculosis (M. tuberculosis) bacillary loads using a guinea pig model. Two formulations were compared: rifampicin encapsulated in poly(lactic-co-glycolic acid) (PLGA/RIF) and PLGA/RIF coated with mycolic acid (PLGA/RIF/MA), against traditional rifampicin treatment and control groups. Data obtained from this study will guide optimisation for future drug efficacy testing, particularly for targeted TB treatment. The guinea pig model was chosen due to its physiological and immunological similarities to human TB infection. Detailed investigations included clinical monitoring, macropathological and histopathological assessments, and bacterial load quantification to evaluate treatment efficacy, side effects, and overall animal health. Key findings indicated differences in survival rates, clinical signs of TB progression, and bacterial load reduction among the treatment groups. The PLGA/RIF group showed promising results in terms of survival rates and bacterial load reduction, suggesting potential benefits of nanoparticle-encapsulated drug formulations. However, the addition of mycolic acid in the PLGA/RIF/MA formulation did not significantly enhance treatment outcomes compared to PLGA/RIF alone, highlighting the complexity of optimizing nanoparticle formulations for TB treatment and the need for further research. The study also addressed the challenges of achieving statistical significance in animal model research, particularly in pilot studies with limited sample sizes and high variability. It emphasized the need for method refinement to lower variability and increase method repeatability and reproducibility, leading to more statistically powered studies to confirm preliminary findings and fully assess the efficacy of nanoparticle-encapsulated TB treatments. Additionally, the study recommended serological testing for TB biomarkers as a method for early TB detection in animal models, potentially enabling earlier treatment initiation and providing insights into infection dynamics and treatment response. Overall, this study laid the groundwork for further exploration of nanoparticle-based drug delivery systems in TB treatment. It underscores the considerations in designing targeted drug delivery, the challenges and potential of PLGA as a drug carrier, and the scope for innovative approaches to improve TB treatment efficacy and patient outcomes. Future studies are encouraged to build on these findings, refine animal models, and explore novel early diagnostic methods to advance TB research and treatment.
Analysis of rabies surveillance in selected African localities and prospects for continent-wide improvements
(University of Pretoria, 2024-10) Nel, Louis Hendrik; Wright, Nicolette; aylamalan@gmail.com; Malan, Ayla Janina-Bertha
Despite being a vaccine-preventable disease, rabies remains a significant public health concern throughout the developing world in particular, including all the countries of mainland Africa, where the disease results in the deaths of more than 25,000 people annually. Through a systematic review of rabies in African countries, a situational analysis highlighted the multifaceted challenges that contribute to the persistence of rabies and the current strategies employed by countries towards rabies elimination. Based on this, six critical barriers to rabies elimination could be identified: high domestic dog populations, inadequate surveillance and reporting, limited access to post-exposure prophylaxis (PEP) and vaccines, economic constraints, cultural factors, and low public awareness. This further highlighted the persistent nature of rabies across most African nations, emphasising the need for effective dog population management strategies and mass dog vaccination campaigns to break the cycle of rabies transmission. Furthermore, inadequate surveillance systems and reporting mechanisms result in a severe underreporting of rabies cases in both humans and animals, leading to an underestimation of the true burden of rabies. As such, national surveillance frameworks need to be implemented and should incorporate multidisciplinary partners and collaborative efforts between neighbouring countries. In an effort to supplement limited surveillance data by predicting gaps in the data, spatio-temporal analyses and data analyses were done using 21 years of laboratory-derived surveillance data from South Africa. The detailed spatio-temporal analysis presented here showed significant disease outbreaks in the eastern half of the country, where the highest human and dog populations could be found. Notably, the study identified underreported districts with limited surveillance, suggesting that enhanced monitoring could reveal larger disease clusters and enable targeted interventions. The analysis also captured the interplay between domestic and wildlife rabies, emphasising the need for integrated control measures. This case study based on South Africa provided evidence of the value of this approach to demonstrate the underestimation of the burden of rabies in the face of poor surveillance data – which is reality for all of Africa. Therefore, replication of this approach in other African countries and regions could add much value to the demonstration of disease burden and the direction of intervention strategies. Finally, considering the de facto challenges of rabies surveillance, a novel rapid diagnostic protocol was designed and evaluated for its potential to provide substantial improvements to the routine diagnosis and surveillance of the disease. In this protocol, the techniques for brain sampling were much simplified and lateral flow devices (LFDs) were implemented in a study that spanned over 28 months on the Unguja island of Zanzibar. Since the LFDs were implemented, a significant increase in the samples collected and sent for diagnostic testing could be seen. This work not only highlighted that LFD devices were highly specific and sensitive in diagnostically screening rabies cases but also showed that their implementation boosted the surveillance network on the island, leading to a more active surveillance approach. This, coupled with real-time reporting, enabled a meaningful increase in both active and passive surveillance across the island of Zanzibar, facilitating rapid outbreak responses, such as targeted and strategic vaccinations to rapidly break transmission cycles before rabies can spread and affect more animals and people. This further highlights the importance of novel techniques to improve overall surveillance.
Functional characterization of candidate Persea americana nucleotide-binding leucine rich repeat (PaNLR) genes during Phytophthora cinnamomi infection
(University of Pretoria, 2024-11) Van den Berg, Noelani; Swart, Velushka; susanna.anbu@up.ac.za; Anbu, Susanna Pearl
Avocado (Persea americana) production is threatened by Phytophthora cinnamomi, the causative agent of Phytophthora root rot (PRR). Current control measures rely on phosphite-based fungicides and partially-resistant rootstocks like Dusa®. However, the immune mechanisms underlying Dusa®'s resistance remain unclear. This study investigated nucleotide-binding leucine-rich repeat (NLR) proteins in Dusa®, which play a critical role in effector-triggered immunity (ETI) and hypersensitive response (HR). Using a Nicotiana benthamiana-P. cinnamomi model system, we identified the biotrophic and necrotrophic phases of infection, applying this knowledge to study avocado NLRs. Six PaNLR candidates were selected based on expression patterns, defense motifs, and effector recognition potential. Transient expression assays revealed that five PaNLRs caused HR only upon pathogen introduction, while one induced cell death independently. Cellular localization studies showed most PaNLRs reside in the nucleus during the resting stage, with one in the cytoplasm. Upon P. cinnamomi infection, all candidates triggered a strong HR, effectively restricting pathogen spread. These findings suggest that PaNLRs adopt diverse strategies to induce immunogenic cell death, balancing resource conservation and rapid defense activation. This study provides the first molecular characterization of PaNLRs, offering insights into avocado immunity and pathways for enhancing resistance in this economically important crop.
The draft genome of the canine parasitic roundworm Spirocerca lupi
(University of Pretoria, 2024) De Waal, Pamela Jean; wiekolize@gmail.com; Rothmann-Meyer, Wiekolize
Spirocerca lupi is a parasitic nematode of dogs and wild canids. It causes spirocercosis which is associated with aortic lesions that occasionally rupture and cause death, as well as oesophageal nodular masses that can undergo neoplasia and malignancy if left untreated. Spirocercosis can be difficult to detect early in the course of the infection and treatment may be ineffectual in later stages of infection. It is endemic to South Africa and is found in most countries but is more prevalent in tropical and subtropical regions where dung beetles serve as intermediate hosts. Identifying new anthelmintic and vaccine targets can lead to new treatments against this parasite. DNA was isolated from two adult S. lupi nematodes. The first was used to sequence the genomic DNA, and the second to sequence the transcriptome. The draft genome of S. lupi consists of 13,627 predicted protein coding genes and is approximately 150 Mb in length. The transcriptome was used to identify potential anthelmintic and vaccine targets. Seven priority anthelmintic targets were identified as well as seven potential vaccine targets along with several known vaccine and immune modulators. In addition to the nuclear genome, the mitochondrial genome was sequenced and compared to the mitochondrial genome of S. lupi sequenced in China. These two genomes were found to be mostly similar. We did identify that the mitochondrial genome is potentially heteroplasmic with cloned sequences of the nad2 gene region from a single adult nematode yielding different gene sequences. The genome of S. lupi was analysed to inform a vaccine that was designed following a reverse vaccinology and immunoinformatics approach. Gene targets were selected using a set of strict criteria. Through this approach eight genes were identified and eight multi-epitope vaccines against S. lupi were designed. Vaccine 6 model 4 was the vaccine that showed the most potential due to its antigenicity, non-toxicity, solubility, non-allergenicity and the more stable 3D structure and binding to both the TLR4/MD2 of the mouse and the domestic dog. This study demonstrates that genome in silico approaches can be used in vaccine target discovery and basic vaccine design although further in vitro and in vivo validation studies are still required to test the full efficacy and effects of any vaccine. This vaccine forms the basis for further study towards a vaccine for S. lupi. With the genome of S. lupi now available there is a vast array of potential future research avenues that can be explored. This includes but is not limited to: improved diagnostic tests, drug and vaccine design, investigation of the biology of the parasite such as the molecular machinery involved in its life cycle, migration through the host, reproduction, immunity, disease and identification of cancer-causing agents.
Proteolysis-targeting chimeras as a novel strategy for targeting epigenetic mechanisms in Plasmodium falciparum parasites
(University of Pretoria, 2024-10-29) Birkholtz, Lyn-Marie; tyrickwelcome2@gmail.com; Welcome, Tyrick
With the continuous emergence and spread of resistance to the only remaining effective antimalarial, artemisinin, novel drugs with novel modes-of-action (MoA) are needed. Current antimalarial efforts aim to identify small-molecule inhibitors that bind to the active site of essential target proteins, thereby killing the causative organism, Plasmodium falciparum. However, such active site-dependent inhibitors face the major challenge of resistance development. We therefore proposed the use of Proteolysis-targeting chimeras (PROTACs) that can induce the degradation of a target protein via the cells ubiquitin-proteasome system rather than inhibiting it. Ideally, for PROTACs to be effective in malaria research, they should bind to essential proteins, such as epigenetic modulators. Here we aimed to evaluate if PROTACs (MZ1 and VZ185) targeting epigenetic mechanisms have activity against P. falciparum parasites and if they are active due to a unique MoA. Both PROTACs were found to be active against the asexual developmental stages of P. falciparum parasites, while VZ185 was found to also be active against the gametocyte stages, implicating multistage activity. The PROTACs additionally showed nanomolar activity against different drug-resistant parasite strains with potentially improved selectivity towards the parasite compared to mammalian cells. We also confirmed that the activity of the PROTACs in the parasite is reliant on the entire molecule and not only its individual ligand components. We also found that the cis-isomers of the PROTACs have improved or similar activity against the parasite, indicating that the interaction with the E3 ligase in the parasite is not stereochemically driven. Co-treatment of the parasites with the PROTACs and a proteasome inhibitor, epoxomicin, did not affect the activity of MZ1 while VZ185 was adversely affected, indicating that the effect of the latter relies on active proteolysis. We determined if the biological action of VZ185 in the parasite was related to epigenetic mechanisms by studying the changes in the morphology of parasites treated with the PROTAC. We found that asexual blood stage parasites treated with VZ185, and cis-VZ185, altered the parasite's ability to produce daughter cells and inhibit haemoglobin metabolism. This study demonstrated that PROTACs could be used as potential antimalarial compounds and a tool to probe parasite biology.
Characterisation of the WAK / WAKL gene family, with a focus on members in Persea americana implicated in defense against Phytophthora cinnamomi
(University of Pretoria, 2024-10-31) Swart, Velushka; Van den Berg, Noelani; aaronharvey0627@gmail.com; Harvey, Aaron Thomas
Phytophthora cinnamomi is a devastating oomycete pathogen, which has a significant negative impact on global avocado (Persea americana) production. The wall associated kinases (WAKs) and wall associated kinase-likes (WAKLs) are pattern recognition receptors which detect fragmented pectin (oligogalacturonides formed during pathogen infection) and activate downstream plant defence responses. The identification and charaterisation of this gene family shows many inconsistencies which needed to be addressed before delving into the avocado WAK/WAKL gene family and describing which member are implicated in the defence response of P. cinnamomi. Thus, this dissertation aimed to critically evaluate the in silico characterization of the WAK and WAKL gene family as a whole and then in avocado, with a particular focus on the genes involved in defence against P. cinnamomi. The study begins with a critical evaluation of current methodologies for identifying and classifying WAK/WAKL genes across various plant species, revealing inconsistencies in identification, classification and characterisation protocols that hinder broader gene family conclusions. The review provides a streamlined approach to help increase the standardisation in this gene family so that all genes defined as WAKs or WAKLs contain the same properties, such as a standardised set of predicted protein domains in the downstream proteins. The review also provides recommendations for characterising the family to allow a broader description to be concatenated for large-scale analyses in the future. The core of the research is the comprehensive in silico characterization of the avocado WAK/WAKL (PaWAK/WAKL) gene family. The study describes the PaWAK/WAKL family composition, gene placement and structure, the protein properties and cellular localisation, phylogenetic relationships, their expression patterns during pathogen infection, and the potential impact of cis-acting elements in targeted regulation during defence. Protein 3D structures and the binding affinities of WAK/WAKLs and the damage-associated molecular pattern, oligogalacturonide, were predicted for defence-implicated proteins. This dissertation examines the PaWAK/WAKL on a genomic, transcriptomic, and proteomic level to identify differences between the partially-resistant Dusa® and susceptible R0.12 rootstocks (while also assessing the partially-resistant LeolaTM) to predict how these factors, in combination, contribute to the increased defence efficiency against P. cinnamomi. These findings highlight the significance of specific PaWAK/WAKL genes in avocado's defence against P. cinnamomi on a multi-omic level. This research not only enhances our understanding of this disease interaction but also serves as a foundation for developing molecular tools to screen for resistant rootstocks for commercial use.
Assessment of groundwater quality for irrigation and drinking purposes in the Limpopo Granulite-Gneiss region, Limpopo Province, South Africa
(University of Pretoria, 2024-09) Claassen, Marius; mirrander.n@gmail.com; Ndhlovu, Nothando Mirrander
The global freshwater availability studies have classified South Africa as water stressed in 1999 and approaching water scarcity by the year 2025. While groundwater is available everywhere, it is not always in suitable quality meaning some groundwater dependent rural communities lack adequate information about the water quality status of their groundwater supplies. While there may be significant groundwater research studies done, the outputs never reach the impacted communities. Groundwater accounts for nearly 70% of rural domestic water supply in Limpopo Province. Furthermore, rapid population growth and more frequent drought events have led to reduced surface water supply and increased in groundwater abstraction, putting groundwater resources under enormous pressure. As communities grow, more groundwater is abstracted, and land use changes to more paved roads, houses, shopping centres, and parking lots, decreasing groundwater recharge. A total of 319 groundwater samples, from 17 monitoring sites located within Limpopo granulite-gneiss region, collected between 2000 and 2017, were analysed in order to evaluate its suitability for drinking and irrigation purposes. The abundance of cations and anions are showing Na+>Ca2+>Mg2+>K and HCO3->Cl->SO42->NO3-+NO2->F-, respectively. The weighted average values show major anions are dominant over the major cations. Na+ accounts for 48% of the cations and HCO3- accounts for 41% of the anions. The pH of groundwater in the study area ranges from 6,9 to 9,2; with over 83,7% of the samples falling within the pH 7,5 to 8,5 range; while 5,0% of the samples are within the pH 6,5 to <7,5 range; and 11,3% samples are within the pH >8,5 to 9,2 range. TDS values range from 107 to 2426 mg/L with a weighted average of 1122 mg/L. At least 57% of the samples are categorically brackish and 43% are fresh. On the Piper diagramme, the water samples are mainly plotting on Ca-Mg-HCO3 type (zone 5) and mixed (Ca-Mg-Cl-SO4 and Ca-Mg-HCO3) type (zone 9), Ca-Mg-HCO3 type indicating carbonate (temporary) hardness. The Gibbs diagramme indicates that groundwater chemistry is controlled mainly by evaporation dominance mechanisms, while high Ca2++Mg2+ concentrations relative to HCO3- concentrations, indicate silicate weathering involving reverse ion exchange. Pearson correlation analysis shows a very strong positive correlation of Na+ with Cl- (0,80), SO42- (0,81), and F- (0,78). Suitability for irrigation assessment results show that all the groundwater samples were suitable for irrigation purposes based on sodium adsorption ratio, residual sodium carbonate, and permeability index. However, for the Kelly ratio index, seven monitoring sites are unsuitable for irrigation purposes. Over 71% of the water samples have concentrations of nitrate higher than the WHO and SANS241 recommended guideline value of ≤11 mg/L, making 15 out of 17 monitoring sites unsuitable for drinking. However, the remaining two sites failed drinking water suitability in TDS, Na, Cl, SO4, and F based on SANS241and WHO, guidelines. To mitigate methemoglobinemia, a simpler, cheaper, and more immediate approach that is within the capabilities of communities is recommended.
In vitro activity and mode of action of the antimicrobial peptide CPF-P2 against Escherichia coli ATCC 700928
(University of Pretoria, 2024-09-16) Gaspar, A.R.M. (Anabella Regina Marques); Bester, Megan J.; Ibrahim, Mohammed Auwal; molebogengonkaetse@gmail.com; Onkaetse, Molebogeng Refemetswe
A major challenge associated with antimicrobial resistance (AMR) is the lack of new drugs especially for the treatment of infections caused by Gram-negative bacteria. Antimicrobial peptides (AMPs) present an alternate solution to the crisis of AMR, as AMPs are novel antimicrobials with unique modes of action and potentially reduced risk of developing antibiotic resistance. Previous studies have identified the antimicrobial potential of the frog skin-derived AMP, the caerulein-precursor fragment-P2 (CPF-P2). To advance the development of this AMP for therapeutic applications, the aim of this study was to further evaluate the antimicrobial activity, mode of action, cytotoxicity and stability against Escherichia coli ATCC 700928. Against E. coli ATCC 700928, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of CPF-P2 was 16 ± 0.7 μg/mL (6.08 ± 0.27 μM), with an MIC/MBC ratio of 1 indicating that this AMP is bactericidal. Against E. coli biofilms, the minimum biofilm prevention concentration (MBPC) of CPF-P2 was 640 μg/mL (243.2 μM), whereas both the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were >2560 μg/mL (> 972.8 μM). This implies that although some biofilm prevention occurred, against established biofilms CPF-P2 was ineffective. Only at 128 μg/mL (48.64 ± 2.64 μM) and 256 μg/mL (97.28 ± 2.25 μM) CPF-P2, did human erythrocyte haemolysis occur while some cytotoxicity was observed against HaCaT cells at 256 μg/mL (97.28 ± 4.81 μM) after 24 hours exposure. The mode of action studies included the determination of the killing kinetics, kinetics of membrane permeabilisation and the subsequent effects on bacteria ultrastructure. At the MBC of CPF-P2 (i.e. 16 μg/mL) the killing time was 120 minutes, mediated by membrane permeabilisation which was time- and concentration-dependent. Ultrastructural studies with scanning electron microscopy, confirmed membrane targeting with structural changes to cell shape, ruptured cell membranes and leakage of cellular content with evidence of blebbing. The stability of CPF-P2 in physiological environments was evaluated. CPF-P2 retained activity in 5% foetal bovine serum (FBS) but lost activity in 25% FBS. Pre-incubation with trypsin also resulted in complete loss of CPF-P2 activity. In conclusion, CPF-P2 was identified as bactericidal, selective for planktonic E. coli with a killing time of 120 minutes at its MBC. The bactericidal activity is mediated by membrane permeabilisation leading to associated changes in cell morphology. Its activity against biofilms was limited and was compromised in physiological environments. Nonetheless, this study identified CPF-P2, as a promising template for the further development of analogues for topical infections.
Diversity and evolution of terpene synthase genes in Ascomycota
(University of Pretoria, 2024-12) Coetzee, Martin Petrus Albertus; Duong, Tuan A.; u15350721@tuks.co.za; Nxumalo, Zinhle Annah-Gloria
Terpene synthase (TPS) genes encode for terpenoids. These enzymes are secondary metabolites with various functions in fungi, such as defense against predators. They are also used in various industries, including the baking, cosmetic, and farming sectors. Despite their biological and industrial importance, little is known about their distribution and evolution in the fungal lineage Ascomycota. The research presented in this dissertation, therefore, investigated their distribution and the evolutionary mechanisms involved in these fungi. The first chapter of this dissertation reviewed the current body of literature regarding the characteristics of TPS genes and their terpenoid enzymes. It also considered the biological roles of these enzymes in fungi and their industrial applications. Finally, the review reflected on bioinformatics methods for analysing TPS genes. Two research chapters then followed the review chapter. Results from the research in these chapters showed that Ascomycota species harbour TPS genes that vary in copy number and type, ranging from two to 56 copies. The type of terpenoids that were identified included prenyltransferase (PT), terpene cyclase (TC) (predominantly sesquiterpenes and diterpenes). Results from the last chapter showed that this variation is due to duplication, gene loss, and horizontal gene transfer (HGT). Ancestral character state reconstruction of the TPS orthologous groups showed that the ancestor of Ascomycota had only three orthologous groups, and the number of orthologous groups expanded and contracted throughout the evolution of the ancestors of extant species.
Effect of large herbivore decomposition on the succession of Mesic-grassland soil microbiomes
(University of Pretoria, 2024-11-19) Cowan, Don A.; Lebre, Pedro Humberto; jacques.fch@gmail.com; Fouché, Jacques
Plant detritus is abundant in grassland but decomposes slowly and is relatively nutrient-poor, whereas animal carcasses are labile and nutrient-rich. Although nutrients from carcasses are highly sought-after, historically, they have been considered insignificant due to their brief decomposition period and minor contribution to the overall landscape nutrition. Recent studies have demonstrated that carcasses significantly alter long-term soil properties at an ecosystem scale. There is a paucity of empirical evidence of the temporal scale of functional and structural succession of the soil microbiome during and after carcass decomposition. Over a period of eighteen months, this study evaluated the functional and structural succession of the soil microbiomes beneath ten Connochaetes taurinus (wildebeest) carcasses. Functional succession was measured by the utilisation of 31 ecologically relevant carbon substrates using BiologTM EcoPlatesTM. Metagenomic analysis of 16S rRNA genes evaluated the bacterial community structural succession. Functional analysis results indicated that most soil microbial processes beneath the carcasses were accelerated for a limited period but resulted in an enduring increase in functional diversity. Substrate utilisation shifted successively and remained evident throughout the study period. Conversely, bacterial diversity was significantly reduced and dissimilar to control soil, although it recovered incrementally to the control soil levels within eighteen months. Biomarkers at different taxonomic levels were identified at various postmortem intervals up to eighteen months

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