Masters Degrees (Microbiology)
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- ItemInvestigating the effects of the plaC accessory protein on disulphide bond formation in plantaricin 423 heterologously expressed in escherichia coli(Stellenbosch : Stellenbosch University, 2024-03) Joos, Carla Grete; Dicks, Leon Milner Theodore; Van Staden, Anton du Preez ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The discovery of antibiotics has revolutionised society. Antibiotics have been used to treat countless diseases and improved the quality of life for many. Unfortunately, the overuse of antibiotics has led to bacteria developing mechanisms to outmanoeuvre the inhibitory effects of these pharmaceuticals. The emergence of antibiotic-resistant bacteria poses a major threat to human health, industries and the economy. Thus, the search for alternatives to antibiotics has become imperative. Lactic acid bacteria (LAB) have a long-standing association with food preservation, pharmaceutical production and use as probiotics. Amongst the myriad of metabolites produced by LAB, ribosomally synthesised peptides referred to as bacteriocins, have gained interest for their antimicrobial properties. LAB secrete bacteriocins to help outcompete other bacteria inhabiting their surrounding environment. This study focused on class IIa bacteriocins, in particular plantaricin 423 isolated from Lactobacillus plantarum 423. Class IIa bacteriocins are pediocin-like and well-known for their antilisterial properties. The production of class IIa bacteriocins has been greatly improved, particularly by their heterologous expression as fusion proteins. In this study, we further improved the heterologous expression of plantaricin 423 through the co-expression with its accessory protein, PlaC, identified in the plantaricin 423-encoding operon. A thioredoxin-fold and CXXC catalytic motif were identified in the PlaC accessory protein, which are characteristics associated with the thioredoxin superfamily. However, thioredoxin-like accessory proteins are uncommon amongst class IIa bacteriocins. An insulin reduction assay based on the dithiol-disulphide oxidoreductase activities of the thioredoxin system was performed and further confirmed that the PlaC accessory protein is a thioredoxin superfamily member. In addition, a peptidase (PEP) domain was identified in the native secretion machinery of plantaricin 423. The PEP domain shares characteristics with C39 cysteine peptidases. The PEP domain was isolated and its ability to proteolytically remove the leader peptide of precursor plantaricin 423 was evaluated. It was hypothesised that the PlaC accessory protein improved the heterologous expression of plantaricin 423 through promoting disulphide bond formation. Previous studies obtained similar results with another class IIa bacteriocin, pediocin PA-1, with its respective accessory protein, PedC. The formation of disulphide bonds in the periplasmic space of Escherichia coli involves two important enzymes, DsbA and DsbC, which function as an oxidase and isomerase, respectively. Unfortunately, disulphide bond formation is poorly understood in Gram-positive bacteria. To improve our current knowledge of disulphide bond formation in secreted proteins from Gram-positive bacteria, the PlaC accessory protein was expressed in E. coli strains with knockout mutations for DsbA or DsbC. In addition, PlaC was heterologously expressed with two plant antimicrobial peptides, VvAMP1 and VvScorpio, that both require four nonconsecutive disulphide bonds. This is the first study that provides experimental evidence confirming that the thioredoxin-like accessory protein, PlaC, identified in the plantaricin 423-encoding operon serves as a DsbC isomerase homologue during the maturation and secretion of plantaricin 423 by promoting correct disulphide bond formation. This is also the first report for a DsbC-like homologue in Firmicutes.
- ItemDiamond based electrodes for water quality applications: disinfection, micropollutant removal and the suppression of biofilm proliferation.(Stellenbosch : Stellenbosch University, 2024-03) Louw, Carli; Wolfaardt, Gideon M. ; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Rivers in most low and middle income countries face a growing burden of pollution in the form of pathogens and persistent micropollutants caused by waste discharge from informal settlements and poorly maintained wastewater treatment plants, operating above capacity. Farming communities are often reliant on the these rivers for irrigation. This study investigated the potential application of diamond based electro-oxidation in decentralized water treatment systems for disinfection and micropollutant abatement. The diamond-electrode based electrochemical in-situ system (DiaDis) forms ozone and hydroxyl radicals via electro-generation. It was hypothesised that the system would possess strong disinfection properties, but that micropollutant abatement would be dependent on the compounds and organic matter present. The disinfection study included the treatment and prevention of biofilms by monitoring the effects on biofilm metabolic activity during DiaDis treatment. A pure culture of Pseudomonas fluorescens sp. was used for the single culture biofilm disinfection study, and for the mixed species biofilm study the culture was sampled from a polluted river. The disinfection capabilities of the DiaDis was found to be comparable or slightly superior to 1:10 dilution sodium hypochlorite in this study for the single culture biofilms, however, the mixed species biofilms were more effectively treated with sodium hypochlorite. Biofilm-forming species have varying resistance to disinfectants and the results suggest the presence of synergistic adaptation. For the single and mixed species biofilms the treatments studied proved more effective when used preventatively than to treat mature biofilms. Removal efficiency of micropollutants by the mixed species biofilms was comparable to published literature, although lower abatement rates were reported for caffeine and acetaminophen, which could be the result of higher organic matter acting as oxidation scavenger in the environmental water. Negative removal rates were obtained for sulfamethoxazole, benzotriazole and efavirenz, likely due to electro- oxidation facilitated reconstitution of breakdown products to the respective parent compounds. The DiaDis was evaluated as a final disinfection step in an aquaponics, as well as a constructed floating wetland system. It performed better in the floating wetland, possibly due to higher levels of organic matter coming from fish-feed in the aquaponics system. Overall the DiaDis showed promising disinfection capabilities, which will benefit from future work to better control pH reduction and to reduce interference of organic matter with micropollutant abatement.
- ItemIsolation and characterization of bacteriophages infecting UTI-associated bacteria and evaluation of phage-derived proteins as potential therapeutic agents and diagnostic probes(Stellenbosch : Stellenbosch University, 2023-12) Aaron, Joshua Alexander; Dicks, Leon Milner Theodore; Perold, W. J. ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Healthcare faces two major problems today, the increased emergence of antimicrobial resistance and the need for rapid diagnostic testing of pathogenic bacteria. The over and or improper prescription of antibiotics has further exacerbated this, also leading to major disruption of the gut microbiome in individuals overcoming diseases. Bacteriophages (phages) can provide the solutions to these current challenges as they solely infect their specific host bacteria. Utilizing whole phages or phage proteins in therapeutics and diagnostics has increased rapidly over the years, offering unique strategies of addressing today’s problems. In this study bacteriophages that specifically target uropathogens were isolated from wastewater treatment plants. Several of these phages were characterized on a genomic and physiological basis. Focus was drawn to a new species of Proteus mirabilis phage belonging to the Novosibovirus genus. The newly identified Proteus_virus_309 was found to drive the emergence of phage insensitive mutants (PIMs). The wild type, phage susceptible, P. mirabilis and phage insensitive mutants were sequenced using the Oxford nanopore sequencing platform which assisted in identifying multiple small nucleotide polymorphisms (SNPs) that may be responsible for the observed phage resistance. Whole genome sequencing of several phages provided an ample source for identifying genes with therapeutic and diagnostic potential tail associated genes from Proteus_virus_309 and Proteus_porphage_301 were selected for protein production and further analysis. Concurrently, previously characterized receptor binding proteins (RBPs) genes from Salmonella bacteriophage vB_SenM-S16 and Staphylococcus aureus phage φ11 were also selected and synthesized. All the selected phage genes were successfully cloned, expressed and the proteins fused with a green fluorescent protein (GFP) were his-tag purified. It was confirmed a putative tail spike protein, TSL309 (ORF57), from Proteus_virus_309 possess depolymerase like activity, evaluated using spot tests, Transmission Electron Microscopy (TEM) and sodium dodecyl sulfate (SDS)-gel analysis of crude capsule extracts. The depolymerase activity observed in TSL309 hampers the biofilm formation and increases de-fouling of Proteus cells on polyvinyl chloride tubing. One aspect of this study shows the potential use of phage derived depolymerases as aiding therapeutics, prompting the requirement for further research into the synergism of phage depolymerases with antibiotics and immune responses in overcoming bacterial infections. The second aspect of the study screened Salmonella and Staphylococcus phage RBPs, Gp38 and Gp45, fused with GFP for their binding capacity toward various bacterial isolates. Bacteria-RBP interactions were evaluated with confocal super-resolution fluorescent microscopy and the fluorescently labelled RBPs. Phage based probes GFP-gp38 and GFP-45 were found to bind to their respective bacterial species, with gp45 binding to a range across Staphylococcus and Enterococcus species. This study highlights a pipeline of identifying, producing, and screening potential phage probes with possible incorporation into a rapid Point of Care (PoC) biosensor device with the aim of providing accurate detection of pathogenic bacteria.
- ItemMicroalgae as a feedstock for ethanol production(Stellenbosch : Stellenbosch University, 2023-12) De Villiers, Dewald; Van Zyl, Willem Heber; Viljoen-Bloom, Marinda; Cripwell, Rosemary Anne; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Microalgae are increasingly considered a source for high-value products in various markets, with applications in the health food, medicinal and industrial sectors. More recently, microalgae have gained interest as a feedstock for biofuel production due to their high starch content. Various studies investigated the challenges of using microalgae in industry, such as low biomass production, complicated harvesting methods and high lipid/carbohydrate content. Although solutions and mitigation strategies have been proposed, the process must be economically feasible to compete with fossil fuels and other biofuel feedstocks. This could be achieved by optimisation of the growth conditions to maximise the biomass and starch yields and/or through enzymatic treatment to release the starch for fermentation to bioethanol. In this study, the growth conditions for two microalgal strains known for their starch - producing capabilities, Chlamydomonas reinhardtii and Chlorella sorokiniana, were optimised for enhanced biomass and starch accumulation, specifically by evaluating different carbon and nitrogen sources. The two strains were grown under mixotrophic conditions, i.e. photosynthesis in the presence of additional carbon sources (glucose and acetic acid). The C. sorokiniana strain displayed the highest biomass production (3.89 g/L) and starch accumulation (0.67 g/L) when grown in Bold Basal Media (10 g/L glucose) with a modified carbon-to-nitrogen ratio (C:N of 8:1). The C. sorokiniana strain was evaluated in a consolidated bioprocessing (CBP) process for starch-ethanol fermentation by optimising the harvest methods and pretreatment options. The study found that enzymatic pretreatment coupled with freeze-drying provided the best results. The C. sorokiniana biomass was pretreated enzymatically with pectinase and xylanase to release the internal starch granules. Consolidated bioprocessing with an amylolytic Saccharomyces cerevisiae strain (co-expressing an α-amylase and glucoamylase) yielded 4.02 g/L ethanol from a 10% microalgal substrate loading at 30°C. This study is one of only a few that observed microalgae growth in a standardised, mixotrophic growth setting where macronutrients were evaluated for optimised starch production.
- ItemCalprotectin (S100A8/A9) as a marker of inflammation and treatment monitoring in cases of juvenile idiopathic arthritis in the Western Cape, South Africa(Stellenbosch : Stellenbosch University, 2023-03) Evert, Christine; Glashoff, Richard H. ; Abraham, Deepthi Raju; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Background: Juvenile Idiopathic Arthritis (JIA) is a common rheumatic disease affecting children and is characterised by persistent inflammation of the joints. The socio-economic climate of South Africa can delay access to treatment to achieve remission. Joint inflammation is currently monitored through C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) measurements. Recent studies have shown that these markers may correlate less well with disease activity than calprotectin. Calprotectin is released by activated monocytes or macrophages at the site of joint damage and binds the TLR4 surface receptor. This protein has also been used to detect subclinical inflammation and may predict risk of relapse. This study aimed to compare standard markers of inflammation with calprotectin, and its relation to other inflammatory markers. Methodology: Blood samples were collected from 22 consented JIA participants. Clinical information and history for each participant was obtained from patient files at Tygerberg Hospital. Monocyte distribution and phenotypic marker expression was investigated using whole blood for surface marker flow cytometry. Plasma levels of calprotectin and JIA associated inflammatory markers (including CCL2, CCL11, CD163, CXCL9, CCL3, CCL22, CD25, CXCL10, IL-1β, MIF, IL12, TNF-α and IFN-γ) were assessed by means of ELISA and Luminex™ multiplex assays. Routine CRP and ESR results were collected from the NHLS TrakCare database. For longitudinal follow up, blood samples were collected from the same cohort 6 months later and assays were repeated. A study database was created with all participant results, at both visits, to investigate relationships between calprotectin and JIA disease activity, as well as the effect of treatment over time. Results: The majority (95%) of participants were already undergoing treatment. Calprotectin was within the normal range for children (127 – 1395 ng/mL) in 86% of baseline samples with a median of 628.6 ng/mL (IQR: 406.2 - 979.8). Only ESR changed significantly (p=0.0079) over time and showed the most evidence for changes in inflammation thereby inspiring analysis by stratification. To evaluate impact of disease phenotype (active vs. remission) and inflammatory state based on ESR expression (high vs. low ESR), participants were stratified into respective groups and compared. Expression of intermediate monocytes at baseline was higher than the expected range (2 - 10 %), with a median of 27 % (IQR: 9 - 43). This distribution is characteristic of inflammatory diseases. Calprotectin correlated significantly (p<0.05) with CRP and ESR at baseline in the returning, remission and high ESR analysis groups. Several notable cases displaying high calprotectin expression were linked to a relapse in disease. Conclusions: Despite limitations, this study confirmed the predictive value of calprotectin in risk of disease relapse and the need for such a marker in the clinical setting to allow for a more tailored approach to treatment. The relationship between intermediate monocyte expansion, calprotectin and disease phenotype also needs to be examined further. Future studies which include treatment naïve participants would be beneficial in assessing the usefulness of calprotectin in monitoring response to treatment.