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

Permanent URI for this collectionhttp://hdl.handle.net/2263/87804

Browse

Now showing 1 - 20 of 207

Unravelling the role of Aurora kinases in cell cycle regulation of malaria parasites
Abrie, Clarissa (University of Pretoria, 2018-11)
The P. falciparum parasite’s life cycle, and implied cell cycle, is strictly controlled allowing the parasite to rapidly develop during the intraerythrocytic stages alternating DNA synthesis and mitosis during endocyclic schizogony. This results in asynchronous nuclear divisions to form multinucleated schizonts during mitosis. However, mitotic nuclear division in P. falciparum parasites remain poorly understood. Several mitotic kinases of the P. falciparum kinome, such as aurora related kinases (ARKs), have been suggested to be cell cycle regulators. The PfARKs are essential for intraerythrocytic stages of the Plasmodium parasite of which PfARK-1 are highly conserved and associated with spindle pole bodies (SPB) during schizogony. The role of PfARK associated with SPB in cell cycle regulation of P. falciparum parasites has been understudied, despite the close link to centrosome and bipolar microtubule dynamics, chromosome segregation and cytokinesis of Aurora kinases in various other organisms. Knowledge gaps regarding the parasite’s cell cycle regulatory mechanisms is mostly due to the complexity associated with cell cycle compartment synchronisation of the parasite under in vitro conditions. In this dissertation, an innovative approach is used for cell cycle compartmentalisation of parasites as a tool to interrogate the importance of PfARK in cell cycle regulation during the intraerythrocytic stages of the parasite through hesperadin-induced PfARK inhibition. Collectively, this dissertation provides extensive insight to PfARK cell cycle regulation during parasite asynchronous nuclear division associated with M-phase progression, chromosomal segregation and spindle formation. The data demonstrate that novel cell cycle regulatory mechanisms of mitotic kinases, such as PfARKs and their involved pathways, serve as attractive future drug targets in Plasmodium parasites.
Anti-biofilm activity of the tick-derived antimicrobial peptide Os(11-22)NH2 against candida albicans ATCC 90028
Chiramba, Court Kudakwashe (University of Pretoria, 2019-02)
The growing occurrence of antimicrobial resistance (AMR) is a global cause for concern due to the decreased availability of effective antimicrobial drugs. Therefore, treating resistant infections can become costly or even impossible. Furthermore, the increase in resistant infections leads to longer hospital stays and higher mortality rates. Resistance is prevalent because microorganisms can form biofilms; communities of cells bound to a surface and covered in an extracellular matrix (ECM), that protects cells from the effects of antimicrobial agents. Besides the ECM, biofilm cells further resist antimicrobial drugs by rapidly developing a number of mechanisms. Therefore, development of novel antimicrobial agents is key to overcoming AMR. One potential alternative to conventional antimicrobial drugs are cationic antimicrobial peptides (AMPs) which are short and amphipathic molecules. Some AMPs possess both anti-planktonic and anti-biofilm activity among other beneficial properties which make them a suitable alternative to conventional antimicrobial drugs. In this study, the anti-biofilm activity of Os(11-22)NH2, a short peptide derived from Os, a derivative of a defensin identified in the tick Ornithodoros savignyi, was investigated. For the purpose of this study, the opportunistic fungal pathogen Candida albicans, one of the leading causes of hospital-acquired infections, was used as the model microorganism. Os was inactive, while the derivative Os(11-22)NH2 was active against planktonic (free-floating) C. albicans with a minimum inhibitory concentration that reduces growth by 50% (MIC50) of 47 μM. The CellTiter Blue (CTB) cell viability assay was used to determine the biofilm inhibiting and eradicating activity. Os(11-22)NH2 inhibited biofilm formation with a minimum concentration of the antifungal agent that reduced biofilm formation by 50% (BIC50) of 81 μM. Inverted light microscopy images confirmed CTB cell viability results and reduced hyphal formation was observed. Treatment of preformed biofilms with Os(11-22)NH2 led to biofilm eradication by Os(11-22)NH2 with a minimum concentration of the antifungal agent that decreased cell viability in a pre-grown biofilm by 50% (BEC50) of 210 μM. Most AMPs target the cell membrane, therefore, membrane permeabilizing activity was investigated using confocal laser scanning microscopy (CLSM) and the DNA binding dyes propidium iodide (PI) and 4',6- diamidino-2-phenylindole (DAPI). Results indicated that cell membranes were permeabilized by treatment with Os(11-22)NH2 during biofilm inhibition and eradication.Some AMPs are known to induce the production of reactive oxygen species (ROS) in microorganisms, leading to cell death. Therefore, the fluorescence producing dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine whether Os(11-22)NH2 induces ROS production. Os(11-22)NH2 induced ROS production during biofilm inhibition and eradication. In the presence of ascorbic acid, a scavenger of ROS, the production of ROS by the peptide was significantly reduced (p < 0.0001). Furthermore, the effect of ascorbic acid on the biofilm inhibiting and eradicating activity of Os(11-22)NH2was determined using the CTB cell viability assay. For biofilm inhibition, no significant difference was observed between treatments in the presence and absence of ascorbic acid, therefore killing during biofilm inhibition was not due to ROS production. For eradication, a significant increase (p < 0.0001) in biofilm eradicating activity was observed in the presence of ascorbic acid indicating that the biofilm eradicating activity of Os(11-22)NH2 was enhanced by ascorbic acid. In conclusion, anti-biofilm activity of Os(11-22)NH2 was determined at micromolar concentrations, indicating a potential antifungal application for this peptide. Future research should reveal its mode of action and combination with other antifungal agents.
Comparative analysis of the blood microbiome in Alzheimer’s disease and healthy individuals
Castillo Vaca, Diego José (University of Pretoria, 2018-12)
Human blood has traditionally been considered a sterile environment. However, evidence in support of a bacterial component in the blood of healthy individuals is steadily accruing. Dysbiosis of this blood-microbiome has been linked to various diseases, i.e., diabetes, cirrhosis and liver- and cardiovascular-disease. Alzheimer’s disease (AD) is an important neurodegenerative condition which, causes dementia in the elderly. According to the pathogen hypothesis, microorganisms may play an important role in the pathogenesis of AD. While the intestinal microbiome could be linked to the onset of AD, the relationship between AD and the blood microbiome remains unexplored. In this study, we performed comparative analyses of the blood microbiome in AD and healthy human individuals, placing an emphasis on AD risk factors and the blood microbial environment, via amplicon and metagenomic sequencing of the 16S rRNA gene. Results indicate that bacterial DNA is present in the blood of both healthy individuals and in AD patients. However, the concentration of bacterial DNA does not differ in Alzheimer’s patients, when compared to healthy participants. Whereas AD patients presented a higher alpha-diversity than healthy participants, beta-diversity did not differ between the groups. Certain haematological factors that represent the blood environment appears to influence bacterial taxonomy and diversity in the blood. Conversely, AD associated risk factors, such as the APOE genotype, does not influence bacterial community structure or taxonomy in the blood of AD patients. Future research should aim to explore the link between the human blood microbiome and the immune system, as well as the relationship between bacterial communities in the blood and those found in the intestinal tract, skin and the oral microbiome.
Epidemiological and molecular approach to determine factors associated with diarrheagenic E. coli in food for children under five in Mozambique
Faife, Sara Lino (University of Pretoria, 2025-05)
Epidemiological and molecular approach to determine factors associated with diarrheagenic E. coli in food for children under five in Mozambique Diarrhoeagenic Escherichia coli (DEC) is associated with diarrhoea and is responsible for around 200,000 deaths worldwide annually, with children under five being the most affected. Children under five from Mozambique are at constant risk for developing diarrhoea due to factors. These include lack of improved drinking water access or sanitation and poor literacy of caregivers, coupled with the fact that their immune systems are under development. In 2018, approximately 500,000 cases of diarrhoea in Mozambique were caused by foodborne pathogens. Recent studies have shown that DEC strains are among the pathogens that cause enteric infection. This study aims to determine sources (food and water) and factors associated with foodborne pathogens causing diarrhoea in children under five years old in rural and urban areas of Maputo province, Mozambique. A total of three hundred children under five years of age with diarrhoea were selected in two Health Care Centres of Maputo province in Mozambique as study cases. The caregivers of the children completed a semi-structured questionnaire. This allowed obtaining information related to demographics, housing conditions, food consumed a week before the children’s diarrhoeagenic episodes, presence of domestic animals, and the general health of the child. Faecal (n =300), food (n = 167) and water (n = 100) samples were collected for diarrhoeagenic bacterial identification. Escherichia coli strains were identified from food and water samples using the Bacteriological Analytical Manual (BAM) protocol developed by the U.S. Food and Drug Administration (FDA). A technical reference guide produced by the Ministério da Saúde de Moçambique (Mozambique’s Ministry of Health) was also used. This analysis was conducted at the Hygiene and Food Technology Laboratory of Eduardo Mondlane University. On the other hand, E. coli strains in faecal samples were identified based on colony morphology characteristics and biochemical tests, performed at the National Institute of Health. The confirmed E. coli strains were whole genome sequenced at the Agricultural Research Council, Pretoria, South Africa. The fastq files of the identified DEC strains were molecularly characterised, and their relatedness was evaluated using the EnteroBase platform. Explanatory variables associated with DEC in children and DEC/Salmonella in food were analysed using chi-square tests and binomial logistic regression. Antimicrobial resistance (AMR) gene profiles were explored through genome analysis using hierarchical clustering, Pearson correlation, and network analysis in R. Only Enteropathogenic E. coli (EPEC) pathotypes (2.0%) were detected in faecal samples of children involved in this study, representing a low prevalence compared to the overall burden of childhood diarrhoea in Mozambique (9.0%). Feeding children with yoghurt was seen as a protective factor against diarrhoea by EPEC. Food and drinking water consumed by the children under five with diarrhoea from Maputo were contaminated with enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and EPEC strains, with a prevalence of 13.0%. Salmonella spp. were also detected, with a prevalence of 2.2%. Feeding children with infant formula, fruit puree, ready-to-eat meals, and bottled water was associated with DEC. There was an abundance of DEC strains belonging to sequence types (STs) ST40 (40.0%), ST206 (20.0%), and ST301 (20.0%) and serotypes O109:H21 (40.0%), O88:H5 (20.0%) and O80:H2. These strains harboured antimicrobial resistance genes conferring resistance to aminoglycosides, fosfomycin, polymyxins, beta-lactam, fluoroquinolone, trimethoprim, tetracycline and sulphonamides. Phylogenetic analysis revealed the relatedness of the DEC strains detected in food, drinking water and childrens’s faecal samples. This suggests that food and drinking water are the potential sources of DEC contributing to diarrhoea in the children under five years old in Maputo. With the present study, we conclude that diarrhoeagenic pathogens were present in faecal samples from children under five with diarrhoea (EPEC) and in food and water they consume (ETEC, EIEC, EPEC and Salmonella spp.). Foods were the main factors associated with these pathogens (yoghurt, infant formula, fruit puree, ready-to-eat meals, and bottled water). The DEC strains belonged to ST and serotypes known to cause diarrhoea, and they carry AMR genes that confer resistance to antimicrobials that are important for clinical practices. Food and drinking water are the potential sources of DEC in children under five years involved in this study. The results of this study provide valuable data on the contribution of food and water to diarrhoea in children, addressing a gap in existing research that has primarily focused on detecting diarrhoeagenic pathogens in faecal samples. In addition, the study demonstrates that DEC strains present in food and water consumed by children in Maputo harbour AMR genes, underscoring the importance of preventing contamination by these pathogens. So, the results of this study highlight the need for education on proper food preparation, storage, and water treatment practices, as well as the adoption of a One Health approach to monitor, prevent, and control the spread of antimicrobial resistance across sectors. The study also underscores the need for further research on caregivers’ attitudes and practices related to food preparation. It also calls for investigation of additional sources contributing to diarrhoea in children under five years old.
Draft assembly of the Acacia mearnsii genome
Xaba, Mondli Audrey (University of Pretoria, 2024-11)
A. mearnsii is an important forestry crop in South Africa occupying 7% of South Africa’s forestry estate. Despite its importance in the wood fibre and tannin industries and the rich history of wattle breeding in the country, there are still limited genomic resources for black wattle. Using high-molecular-weight DNA extraction methodology and data produced on a state-of-the-art long-read sequencing platform (Oxford Nanopore Technologies PromethION), we assessed the performance of three long-read genome assemblers for high-quality draft genome assembly of black wattle. We obtained 160 Gbp of sequencing data on a single PromethION flow cell, which amounts to 230X coverage of the black wattle genome (assuming a genome size of 686 Mbp). We produced a draft genome assembly comprising 430 contigs, with a contig N50 value of 2.3 Mbp, a total assembly size of 686 Mbp and a BUSCO (plant conserved single copy orthologs) completion score of 96.9%. De novo gene annotation identified 51,190 genes in the genome, with 62% of the genome marked as repeat content. We used single copy orthologs and a complete chloroplast genome assembly to assess the phylogenetic history of black wattle in relation to other plant taxa. Using in silico approaches, we have also identified microsatellite DNA loci and used these for the development of oligonucleotide primers for DNA fingerprinting and parentage analyses in wattle breeding. This study demonstrated the efficiency of long-read DNA sequencing technology and current genome assemblers in producing a high-quality de novo draft genome assembly for A. mearnsii, which will serve as a reference for future studies of genome biology and genetic dissection of agronomic traits in black wattle.
Evaluation of the in vitro antiplasmodial and toxicity profiles of novel drug delivery formulations for combination therapies
Muruya, Martha Joy Oguttu (University of Pretoria, 2024-11)
While considerable progress has been made towards malaria elimination over the past several decades, it continues to inflict a heavy burden on socio-economically challenged countries. Artemisinin-based combination therapies, including a fast-acting artemisinin component such as dihydroartemisinin (DHA), have been highly effective in slowing down the rate of drug resistance development and remain the mainstay of control against the disease. However, the complexity of the P. falciparum life cycle combined with antimalarial resistance development has necessitated the development of alternative therapeutic options. As malaria control interventions move toward disease elimination, the focus has shifted towards developing compounds targeting transmissible, gametocyte stages, which remain in the human host long after the asexual stages are cleared. While the clinical candidate MMV390048 exhibits multistage antimalarial activity, inefficient pharmacokinetic and pharmacodynamic properties, poor patient compliance, and the requirement for long-term drug administration pose a challenge for developing dual-stage therapies incorporating this compound. Here, the encapsulation of MMV390048 with the known clinically active DHA in a slow release, bio-functional 3D polymer drug delivery system using P. falciparum as a model organism, is interrogated for its ability to deliver novel combinations with therapeutic applications. This drug delivery system uses DHA- and MMV390048-loaded solid lipid nanoparticles (SLNs). SLNs can directly release DHA and MMV390048, ensuring an antimalarial therapeutic effect. The in vitro inhibitory activity of a novel, dual active drug combination, DHA and MMV390048, was evaluated against asexual blood stages and transmissible gametocyte stages. Evidence for the combinatorial treatment of asexual and gametocyte stages with DHA and MMV390048 is provided. This revealed that late-stage gametocytes are susceptible to inhibition by the combination, an attractive point for transmission-blocking treatment geared towards elimination. DHA- and MMV390048-loaded SLNs did not retain antimalarial activity compared to the unencapsulated drugs against asexual stages, with excessive haemolytic effects and cytotoxicity. Ultimately, this project presents informative and preliminary evidence of polymer drug delivery systems for administering antimalarials. This strategy may contribute to improved pharmacokinetic and pharmacodynamic properties of transmission-blocking drugs and eventual malaria elimination.
An ecological approach to risk characterisation of antimicrobial resistance from South African urban wastewater and freshwater microbiomes
Makumbi, John Paul (University of Pretoria, 2025-05)
Antimicrobial resistance (AMR) is routinely called ‘the silent pandemic’ despite the substantial impact on public health. Natural and engineered environmental systems such as rivers and wastewater treatment plants (WWTPs), may be AMR reservoirs and hotspots. However, we lack comprehensive insights regarding the role of these ecosystems in environmental AMR persistence and dissemination. There is a substantial lack of data on the key ecological parameters that influence AMR dynamics as well as the primary proxies necessary for evaluating dissemination risks. Notably, the potential role of extracellular DNA (exDNA), in facilitating the persistence of antimicrobial resistance genes (ARGs), remains underexplored. This study combined metagenomics to identify antimicrobial resistant bacteria (ARB) and ARGs, with chemical analyses, including liquid chromatography-mass spectrometry (LC-MS) and in-situ measurements, to assess resistome persistence and transition in nine urban WWTPs and associated rivers in Tshwane, South Africa. The findings showed that WWTPs are ineffective at removing high-risk ARGs, including beta-lactamase (bla), aminoglycoside (aac, aad, ant, aph) and mobile colistin resistance (mcr) genes, which are associated with resistance to critically important antibiotics, with effluents showing higher ARG abundance (p ≤ 0.05) than influents. This resulted in the dissemination of ARGs and ARB into rivers, leading to higher ARG occurrences at downstream sites compared to upstream. Environmental compartmentalisation between the water column and sediment microenvironments influenced AMR persistence and dissemination in the receiving rivers. River pelagic zones appear to be crucial for the spread of pathogenic ARB, while sediments were major reservoirs for ARGs. Physicochemical factors, including temperature, pH, nutrients and heavy metal contaminants, substantially influenced ARG distribution. The findings support the assertion that physicochemical factors may be valid proxies for AMR surveillance and risk assessment. Analysis of exDNA demonstrated its underestimated role in shaping AMR patterns in these aquatic systems. To our knowledge, this is the first study to investigate exDNA-associated AMR in an African wastewater and freshwater system. exDNA predominantly carried several high-risk ARGs. These data suggest potential increased risk of AMR persistence in effluents and rivers, with 66% of the WWTPs showing higher ARG counts in effluents compared with influents. The exDNA resistome also suggests high horizontal gene transfer (HGT) potential, driven by mobile ARGs on plasmids and bacteriophages. Notably, WWTPs using combined activated sludge process (ASP) and biofilter (ASP-biofilter) treatment methods showed reduced ARG diversity in the effluents compared to those using ASP-only treatment. However, the differences in ARG abundance between influents and effluents were not statistically significant (p = 0.05). This finding suggests that WWTPs employing additional treatment stages beyond conventional ASP may help reduce AMR dissemination risks. This study highlights the potential benefits of incorporating ecosystem-based approaches into AMR control practices. Specifically, improving ARG removal efficacy at WWTPs is as crucial as reducing antimicrobial-resistant bacterial load. This may be achieved through the implementation of additional barriers such as biofiltration. More broadly, the findings from this thesis emphasise the importance of integrated frameworks to monitor how environmental stressors and anthropogenic influence shape microbial evolution, particularly in pathogenic species that pose a public health risk.
Taxonomy and comparative genomics of bacterial species associated with onion bulb rot focusing on Rahnella species
Mnguni, Fanele Cabangile (University of Pretoria, 2025-02)
Onions (Allium cepa L.) are one of the most widely consumed vegetables globally, thus playing a significant role in food security. However, onion bulbs are highly susceptible to bacterial pathogens, and emerging opportunistic pathogens such as Rahnella species, which have become notable threats to onion production. Identifying these pathogens is essential for managing disease outbreaks. In Chapter 1, we conducted a literature review focusing on the cultivation of onion bulbs and then explored onion production in South Africa and the United States. In addition, a number of onion bacterial pathogens were discussed that included Burkholderia, Rahnella, Erwinia, Serratia, Enterobacter species, the Pantoea complex, and Klebsiella. The management strategies currently implemented to help combat onion diseases, both pre- and post-harvest, were also reviewed. To understand the underlying issues related to bacterial diseases in onions, we analysed the pathogenicity factors commonly utilised by these pathogens. This analysis enhanced our understanding of the bacterial taxonomy of onion pathogens, pathogenicity mechanisms, and genomics, which is crucial for identification of pathogenicity traits used by pathogenic bacteria. In Chapter 2, bacterial pathogens responsible for onion bulb rot in South Africa during the 2021–2022 period were characterised and identified. Diseased onion samples were collected from fresh produce markets and storage facilities in the Western Cape, Free State, and Gauteng provinces. Symptoms observed included softened necks, scale dryness, internal discolouration, slippery, and sour skin. Pathogen identification revealed the presence of Burkholderia gladioli, Klebsiella grimontii, Klebsiella michiganensis, Klebsiella pneumoniae, Pantoea eucalypti, and a Pectobacterium? sp. Koch’s postulates confirmed that all these bacteria were causal agents of onion bulb rot. This study emphasised the importance of identifying bacterial pathogens to reduce pre- and post-harvest losses and sustain onion production in South Africa. In Chapter 3, Rahnella species isolated from onion bulb rot in South Africa (SA) and the United States (USA) were identified using multilocus sequence analysis (MLSA) based on four housekeeping genes (atpD, gyrB, infB, and rpoB. The isolates clustered into five species, viz. R. perminowiae, R. aquatilis, R. aceris, R. variigena, and R. victoriana. Among them, R. perminowiae, R. aquatilis, and R. aceris were the the most frequently isolated. Pathogenicity testing revealed that Rahnella isolates caused symptoms on onion bulbs but did not affect onion leaves and were negative in the red scale necrosis (RSN) assays. Geographical location did not appear to influence the diversity or pathogenicity of these isolates. This analysis highlights the growing importance of Rahnella species as emerging pathogens in agriculture and there is a need to understand their opportunistic tendencies in onion bulbs. In Chapter 4, comparative genomic analyses were used to investigate pathogenicity-related traits in five Rahnella strains isolated from onion bulbs: R. aquatilis (strains 20CA0197 and 20CA0198), R. aceris (samples 20WA0051 and 20WA0057), and R. perminowiae FS4. The pathogenicity-related genes were categorized into metabolic functions, secretion systems, motility, adherence, and lipopolysaccharide biosynthesis, in an attempt to highlight their potential roles in plant interactions. The presence of the thiosulfinate gene clusters in R. aquatilis (strains 20CA0197 and 20CA0198) and R. perminowiae FS4 suggests an adaptation to thiosulfinate-rich environments, in contrast, R. aceris (strains 20WA0051 and 20WA0057) did not possess thiosulfinate gene clusters. The copper resistance genes were absent in all five Rahnella strains. Additionally, biosynthetic gene clusters related to siderophores, quorum sensing, and antimicrobial compounds were identified. Further functional validation is necessary to determine the precise roles of these genes in plant-bacteria interactions and to assess whether they contribute to Rahnella's opportunistic behaviour in onion bulbs. This study reports on the growing threat of opportunistic bacterial pathogens to onion production and highlights the need for comprehensive disease monitoring strategies for opportunistic bacteria. The identification of diverse bacterial species, including Rahnella, Burkholderia, Pantoea, Klebsiella, and Ewingella allii, provides new insights into the complexity of onion bulb rot and emerging opportunistic pathogens. Comparative genomic analyses revealed critical pathogenicity-related traits, indicating potential adaptation mechanisms to onion-associated environments. While Rahnella species exhibited mild pathogenicity, their increasing prevalence necessitates further research to assess their long-term impact on onion crops. Future efforts should focus on pathogen surveillance, functional validation of virulence factors, and the development of sustainable control measures to safeguard onion production and food security.
Investigating the molecular mechanisms underlying avocado sunblotch disease
Joubert, Melissa (University of Pretoria, 2025-05)
Avocado sunblotch disease is recognised as a major global concern for avocado production, and has a significant impact on the South African avocado industry. The disease is marked by the appearance of distinct chlorotic symptoms on fruit, stems and/or leaves of affected trees. These symptoms are triggered after infection by avocado sunblotch viroid (ASBVd) - a small, single-stranded molecule of RNA which does not encode any proteins. Importantly, infection by ASBVd may remain asymptomatic for long periods of time, though avocado trees without chlorotic symptoms have been known to display altered growth habits and decreased fruit yield. As yet, there have been no studies into the molecular mechanisms underlying the subtle phenotypic changes observed in asymptomatic ASBVd-infected avocado, and the process by which the viroid elicits chlorosis in symptomatic interactions remains unknown. Investigations into other viroid-host interactions have explored how the host transcriptome changes upon viroid infection, and how cleavage of viroids by host RNA silencing machinery may have inadvertent downstream effects on the accumulation of host transcripts, which can lead to symptom development. The aim of this work was therefore to elucidate processes involved in ASBVd pathogenesis using next-generation sequencing (NGS) to explore the host transcriptome in asymptomatic ASBVd-infected avocado, as well as to investigate whether viroid-derived small RNAs (vd-sRNAs) produced from ASBVd can be implicated in the triggering of leaf chlorosis in symptomatic infections. Analysis of the host transcriptome in six asymptomatic ASBVd-infected avocado nursery trees revealed notable changes in host gene expression in symptomless carrier trees. Asymptomatic infection by ASBVd resulted in the differential expression of over 600 avocado genes with roles in several important plant processes. Significantly affected host pathways included plant immune responses, hormone signalling networks, transcription regulation, production of secondary metabolites, transport of cellular compounds and processing of proteins. Observations of transcriptional changes in ASBVd-infected trees without chlorosis indicated that latent viroid infection influences molecular pathways in avocados, even when sunblotch symptoms are absent. This research was the first to examine the impact of ASBVd infection on global gene expression in avocado, highlighting important pathways in this plant-pathogen interaction which can be explored in depth in future investigations. To elucidate molecular mechanisms responsible for manifestation of sunblotch symptoms, we examined the viroid sequence variants and vd-sRNAs present in bleached and asymptomatic leaf tissues from symptomatic avocado trees. The mutation within the ASBVd genome associated with chlorotic symptoms was confirmed to be the addition of a uracil residue within positions 115-118 of the genome. ASBVd variants carrying this pathogenic determinant accumulated to different levels in green and yellow leaf tissues of symptomatic trees. The elevated viroid load in bleached tissues resulted in the production of copious ASBVd-derived small RNAs (ASBVd-sRNAs), which were significantly more abundant in yellow tissues when compared to green tissues. ASBVd-sRNAs containing the pathogenic determinant associated with chlorosis were limited almost entirely to yellow tissues – these were predicted to target many avocado genes for silencing. Twenty-five of the putative targets were shown by transcriptome analysis to be downregulated in yellow tissues relative to green tissues. One of the suppressed genes had strong evidence for RNA silencing guided by ASBVd-sRNAs, and its corresponding protein is predicted to function in avocado chloroplast pathways. The silencing of this gene in yellow tissues suggested that its downregulation might have a role in triggering the bleaching of leaves in ASBVd-infected avocado. This study was the first to employ NGS to examine the ASBVd-sRNAs associated with bleached leaves of sunblotch-affected trees, and represents the earliest exploration of putative avocado genes whose silencing by ASBVd-sRNAs may be involved in the elicitation of bleaching symptoms in avocado sunblotch disease. The findings presented in this thesis signify the first recent exploration of the ASBVd-avocado interaction. Collectively, the results of our investigation start to shed light on the molecular mechanisms involved in ASBVd pathogenesis. This work can be built on in future studies to present an even more comprehensive understanding of how ASBVd infection affects the avocado host and triggers avocado sunblotch disease.
Comparative immunogenicity of 3 recombinant Adenovirus-based immunocontraceptive vaccines with different hormone targets
Nel, Carla (University of Pretoria, 2025-02-28)
The ever-expanding stray dog population is a major concern in many developing countries. Stray dogs are unfairly exposed to inadequate living conditions, starvation, neglect and harassment. They also burden an area by lowering its sanitation, safety and appeal. Many diseases (such as rabies) are spread by stray dogs, endangering humans and other animals. This highlights the importance of dog population management. There are many different forms of dog population management but immunocontraceptive vaccines seem to be the most promising in terms of cost, resource efficiency, practicality and targeting the source of the problem. In this project, two different immunocontraceptive vaccines (AdGnRH-TT and AdKP-TT), each expressing a different reproductive hormone target (kisspeptin or GnRH) couped to a partial tetanus toxin gene to enhance immunogenicity, were developed. Previously our research group constructed a dual hormone target immunocontraceptive vaccine (AdGKT) which contained the kisspeptin, GnRH and partial tetanus toxin genes. All three of these vaccines were administered to mice and the anti-hormone antibody titres quantified to determine which vaccine was the most immunogenic and should be used as the candidate vaccine to test in the target species, namely domestic dogs. It was found that the AdGKT vaccine was by far the most immunogenic candidate, yielding far greater, statistically significant anti-hormone antibody titres compared to the two single hormone target vaccines, which in fact led to no seroconversion in mice. Therefore, the AdGKT vaccine will be used in future trials involving dogs.
Novel analogues of the scorpion venom-derived peptide, opistoporin 1, targeting gram-negative bacteria
Van der Walt, Mandelie (University of Pretoria, 2024-11)
Antimicrobial peptides (AMPs) are promising candidates and lead compounds for the development of next generation antimicrobials. At the onset of this study, 46 AMP candidates, including 27 novel or with unknown activity, were identified from African species such as scorpions, frogs, ticks, and primates. Antimicrobial screening against a diverse panel of susceptible and resistant Gram-negative and Gram-positive bacterial strains as well as fungi was undertaken. The results revealed that while many of the AMPs exhibited potent antimicrobial activity, significant cytotoxicity towards host cells, a common challenge in AMP therapeutic development, was also identified. To address this, multivariate analyses were employed to correlate 28 physicochemical peptide properties to the antimicrobial efficacy and cytotoxicity of the 46 AMPs, aiming to identify characteristics that drive selective antimicrobial effectiveness while minimising toxicity. Principal component analysis (PCA) and quantitative structure-activity relationships (QSAR) revealed that an increase in lipophilicity, hydrophobicity, or residue side chain surface area results in increased activity as well as toxicity of an AMP. Crucially, the total negative lipophilicity contributed by polar residues (Lp) was identified as the key physicochemical property determining selective antimicrobial activity. The scorpion derived peptides proved particularly effective against Gram-negative bacteria. Among them, Opis16a emerged as a novel peptide with significant potential for therapeutic development. Subsequently, a more detailed assessment of the activity, stability, and mechanism of action of Opis16a, particularly against sensitive strains and drug-resistant Gram-negative clinical isolates, was undertaken. Findings were that Opis16a effectively penetrates the intricate outer membrane of Gram-negative bacteria, leading to rapid bacterial cell death. Mechanism of action studies, conducted with a standard laboratory Escherichia coli ATCC 700928 strain and a multidrug-resistant A. baumannii NICD 15283 clinical strain, revealed that Opis16a binds to lipopolysaccharides, destabilises the outer membrane, depolarises the inner membrane and finally induces lethal permeabilisation of the cytoplasmic membrane. The strong bacterial selectivity and retained activity in serum, further supported the therapeutic potential of Opis16a for topical applications. Consequently, activity was further assessed in the in vivo Galleria mellonella model of A. baumannii burn wound infection. Treatment with Opis16a results in a three-fold improvement in larval survival compared to untreated A. baumannii-infected larvae, with an efficacy similar to polymyxin B. Consequently, Opis16a emerges as a novel membrane-destabilising AMP showing promising in vitro and in vivo efficacy against Gram-negative bacteria, suggesting potential for advancement in topical applications. To further increase the activity of Opis16a through rational design, four novel analogues were designed with increased cationic charge where an Asp residue in position five was replaced with a Lys (Opis16aDtoK) or where Lys residues at various positions in the sequence were replaced with Arg (Opis16aCterKtoR, Opis16aNterKtoR and Opis16aAllKtoR). Molecular dynamics (MD) simulations analyses revealed an increase in hydrogen bonding between the analogues and Gram-negative membrane lipids, along with increased membrane insertion in silico. Computational findings were validated through experimental in vitro screenings against a panel of Gram-negative bacteria, including standard laboratory and drug-resistant clinical strains. The analogues showed improvement in activity up to 16-fold compared with the parent Opis16a peptide, while maintaining strong selectivity for bacteria over HaCat cells (human keratinocyte cell-line). The analogue with the greatest improvement in activity and selectivity, Opis16aCterKtoR, also showed increased membrane permeability and in vivo protection of larvae infected with a multidrug-resistant E. cloacae NICD 16103 clinical isolate. In summary, novel structural characteristics for the design of AMPs were developed, and subsequently Opis16a and Opis16aCterKtoR were discovered as novel AMPs with promising therapeutic potential for the treatment of Gram-negative topical infections.
Sporothrix schenckii and its possible origins in South Africa
Fuchs, Taygen (University of Pretoria, 2025-02)
Sporothrix is a genus of dimorphic fungi primarily associated with environmental habitats, though several species also cause infections in mammals. This subcutaneous infection is typically transmitted through trauma to the skin and can lead to chronic and debilitating disease. The global health implications of Sporothrix are significant, with the disease increasingly recognised as an emerging concern, particularly due to the current epidemic in South America. In South Africa, sporotrichosis holds a unique epidemiological significance, with large outbreaks in the gold mining industry previously recorded. Despite the epidemiological significance of pathogenic Sporothrix in South Africa, little is known about the species diversity and genetic variation of these pathogens in the country. This study aimed to address key ecological and epidemiological questions, particularly focusing on elucidating the environmental ecology and diversity of these pathogens in South Africa. Investigation into the presence of Sporothrix in samples collected from gold mine substrates, revealed an untapped reservoir of biodiversity in the gold mine environment. Recently developed molecular markers were applied to a population of Sporothrix species isolated from clinical samples from across South Africa. This revealed surprising species and genetic diversity among the clinical isolates, further allowing for the distribution of Sporothrix lineages across the country to be elucidated.
Population structure and diversity of two pine needle pathogens in Colombia
Piso, Anja (University of Pretoria, 2025-02)
Planting Pinus species has contributed significantly to plantation forestry worldwide. Although mostly native to the Northern Hemisphere, pines have become established in most Southern Hemisphere countries due to their adaptability to almost all climatic conditions and various soil types. Their introduction into countries beyond their native habitats has inadvertently led to the anthropogenic introduction of pathogens into these non-native ranges. This dissertation focuses on two pine needle pathogens present in the Colombian pine plantations, namely Lecanosticta pharomachri and Dothistroma septosporum. A set of microsatellite and mating type markers was developed for Lecanosticta pharomachri and used to screen 120 isolates. The population genetics structure and diversity were then investigated. Existing microsatellite and mating type markers for Dothistroma septosporum were utilised in the investigation of the population structure and diversity of the pathogen which is now infecting trees which were previously thought to be tolerant to the disease.
Genome sequences and molecular resources for Macadamia tree breeding in South Africa
Ranketse, Mary (University of Pretoria, 2024-11)
Breeding for improved tree species is a long and tedious process due to the long generation time, and plants typically have complex genomes and varying phenotypes. Breeding programs require many factors to be considered to firstly establish and then maintain the objective of breeding for improved cultivars for various environments. The application of DNA based molecular markers have shown to be very useful at reducing the breeding cycle time and the efficient management of breeding programs and genetic resources. Microsatellite markers or simple sequence repeats are a low cost, rapid DNA marker system that is efficient for various analyses such as cultivar identification, population diversity and parentage. Whole genome sequencing and assembly is a powerful tool for understanding and characterising genomes, and for the discovery of DNA based molecular markers. Whole genome sequencing is the first step towards unravelling the complex plant populations and genomes. Genome annotation is the next step that adds a layer of biological information that can be used to understand complex biological processes and varying phenotypes. Macadamia nuts are the most expensive in the world and the genus is the most economically important in the Proteaceae family. South Africa is the largest producer of macadamia nuts globally and is thus an important crop to the country, although Macadamia species is native to Australia, and was commercialized in Hawaii. Due to its importance, this study aimed to understand the population dynamics of Macadamia in South Africa using microsatellite markers; and conduct whole genome sequencing, assembly, and annotation of important cultivars to contribute towards developing molecular information that can be used for breeding programs. The key findings of this study are presented below. The South African macadamia industry mainly grows cultivars that are imported from various countries. Thirteen microsatellite markers were used to perform genetic fingerprinting, determine the genetic diversity and population structure of 110 macadamia cultivars in South Africa, in the context of international genetic diversity. The present study compared 31 locally selected cultivars to 31 imported from Hawaii, 19 from Australia, two from California, one from Israel, and 26 from a local breeding population. The microsatellite markers were able to differentiate the two commercial species Macadamia integrifolia and Macadamia tetraphylla into separate groups and the two groups coincided with countries of origin. The South African local selections were mainly composed of M. tetraphylla like cultivars. The Hawaiian imported selections were spread over the M. integrifolia like species group, and a second group that was intermediary of M. integrifolia and M. tetraphylla, consisting of hybrids of varying degrees between the two species. The Australian selections were mainly in the hybrid range, with a few accessions in the M. integrifolia like group. The results showed that the local South African macadamia selections had a unique genetic structure compared to the Hawaiian and Australian selected cultivars. We sequenced, assembled, and annotated three cultivars using Illumina short read sequencing and Oxford Nanopore long read sequencing. Santa Anna is an M. tetraphylla species representative, and two hybrid cultivars of importance to South Africa, Beaumont/HAES 695 (M. integrifolia x M. tetraphylla) and HAES 791 (M. integrifolia x M. tetraphylla x M. ternifolia). The genome assembly sizes ranged from 750.0 Mb for Santa Anna, 762.2 Mb for HAES 695 and 836.5 Mb for the HAES 791 cultivar. Santa Anna had the least number of contigs (579), and the HAES 695 and Santa Anna genomes had 705 and 965 contigs respectively. Contig N50 for HAES 695 and Santa Anna were 2.1 Mb and 1.9 Mb, and HAES 791 was 3.6 Mb. The BUSCO completeness scores were 97.0% and 97.4% (HAES 695 and Santa Anna) and 99.0% (HAES 791). Genome annotation resulted in 37,572; 36,328; and 30,600 genes found in Santa Anna; HAES 695; and HAES 791 respectively. The genome assemblies were compared to the published M. integrifolia HAES 741, HAES 344 and GR1, and an M. tetraphylla genome. Our genome assembly and annotation statistics are comparable to the published genomes and are contiguous and of high quality. Nut oil is an important trait in macadamia as macadamia has the highest content of healthy fatty acids, specifically palmitoleic acid (omega 7 fatty acid), which is not found in concentrations higher than 1% in other tree nuts. This study analysed the fatty acid biosynthesis associated genes from the annotation data and compared our results to other tree nut and oil producing crop species. Four protein families important to fatty acid biosynthesis were analysed: fatty acid desaturase (FAD), stearoyl-CoA-desaturase (SAD), 3-oxoacyl-acyl-protein-carrier-synthase (KAS), and the oleoyl-acyl carrier protein synthase (FATA) and palmitoyl-acyl carrier protein synthase (FATB). The results revealed that Macadamia had the most stearoyl-[acyl-carrier-protein] 9-desaturase 6 (SAD6) encoding genes, followed by palmitoyl-acyl carrier protein thioesterase (FATB) encoding genes, compared to the other species. Macadamia had more KAS encoding genes compared to other tree nut species. The study was able to determine the unique genetic profile of South African locally selected cultivars and developed a technology pipeline for the local macadamia nut industry to perform routine genotyping analysis using microsatellite markers. Furthermore, the genome assemblies and annotation will add to the growing genomic resources for Macadamia. The fatty acid biosynthesis associated gene analysis may explain the unique fatty acid content of macadamia nuts and deserves further investigation beyond this study. In conclusion, this study forms an important foundational analysis of Macadamia genomics and contributes towards developing molecular tools towards advanced genomic breeding programs for South Africa and globally.
Mitochondrial genomes from the Ceratocystidaceae and species boundaries in Ceratocystis
Viljoen, Anien (University of Pretoria, 2024-11)
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
Strydom, Hannes (University of Pretoria, 2024-11)
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
Venter, Natanya (University of Pretoria, 2024-10)
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
Voges, Suzelle (University of Pretoria, 2024-11)
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
Muzondiwa, Dillon (University of Pretoria, 2025-02)
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
Watson, Savannah Jade (University of Pretoria, 2024)
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.

Browse

Recent Submissions