Electronic Theses and Dissertations (Masters)

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A Clot to Uncover: FOXP3 and SARS-CoV-2 Nucleocapsid Interactions and Their Effect on DNA Binding
(University of the Witwatersrand, Johannesburg, 2024) Mcinnes, Keiran; Fanucchi, Sylvia
During COVID-19, systemic coagulopathy, which can lead to strokes and embolisms, is often observed in COVID-19 patients and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that leads to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development and loss of FOXP3 function leads to platelets that resemble those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV- 2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and is potentially able to alter regulation of host protein expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS CoV-2 NC may provide new clues as to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for potential interactions in the absence and presence of DNA using pull-down assays and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA. Additionally, it was found that both proteins are capable of binding to DNA at the same time, but excess NC was found to cause FHD dissociation from the FHD- NC-DNA complex. This result implicates NC in FOXP3 dysfunction which may be associated with the coagulopathy and other symptoms seen during COVID-19. Additionally, NC DNA binding does not appear to be driven by the FOXP3 consensus sequence, indicating that FOXP3 may not be the only transcription factor potentially dysregulated by NC
Analysing RNA-sequence data for pancreatic ductal adenocarcinoma tissue samples to identify potential biomarkers
(University of the Witwatersrand, Johannesburg, 2023-09) Jamal, Khadija Sanober; Kaur, Mandeep
Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of pancreatic cancer and is the fourth leading cause of death with a five-year survival rate of less than 10%. Patients are asymptomatic until detection is observed at a metastatic stage, hence contributing massively towards the high mortality rate. This study was conducted to explore PDAC and its two main subtypes, the classical and basal-like subtype, in an in-depth level via bioinformatic analysis. Bioinformatics is a computational approach to evaluate biological data by analysing omics data including genomic expression and proteomic sequences. A workflow consisting of programmes and web-tools was used to analyse PDAC RNA-sequence data. The sample sets were grouped according to tumour, stage, and subtype. The workflow began with quality control using FastQC and Trimmomatic. Alignment of sequencing files and counts were done through HISAT2 and HTSeq. The main component of this workflow was differential gene expression analysis to identify differentially expressed genes (DEGs), statistically significant genes, per compared conditions. WGCNA was used for co-expression analysis to identify the hub genes involved in regulating the biological network. Lastly, in-silico validation was done by using available web tools to support the findings of this workflow. The identified tumour genes included S100A11, PKM, GPRC5A, LAMC2 and ITGA2, which may represent as universal biomarkers as sample extraction was performed from data generated from individuals belonging to 8 different countries. KRT13 and IL6 were identified in the advanced stage and their role in cancer progression have been explored in this current study. The basal-like subtype had CAV1, DCVLD2 and TGFB2 genes that contribute to treatment resistance. The common dysregulated genes in the basal-like subtype and advanced stage were analysed to evaluate the link between subtype and stage which included WNT3A, TP63, KRT13 and IGF2BP. Coexpression analysis revealed hub genes for tumour (KIF4A, SPAG5, RRM2 and AURKA), basal-like subtype (BUB1, DEPDC1 and KIF14) and classical-subtype (PTPRN and CAMK2B). Through a machine learning model, recall, precision and accuracy scores per sample conditions for the DEGs were all above 94%. These potential biomarkers all have significant roles in promoting cancer progression, aggression and resistance. Hence, these may serve as a less invasive screening method for PDAC as DEGs were classified based on tissue or blood (extracellular vesicle) biomarkers. However, further wet laboratory validation is required for these biomarkers.
Differential expression analysis of PMA and 1,25(OH)2D3-induced monocyte-to-macrophage differentiation in THP-1 cells
(University of the Witwatersrand, Johannesburg, 2023-09) Perumal, Kelda Chloe; Meyer, Vanessa; Gentle, Nikki
The process of monocyte-to-macrophage differentiation is studied in vitro through the use of promonocytic model cell lines, such as the THP-1 cell line, where commonly used differentiation inducing agents include phorbol-12-myristate-13-acetate (PMA) and the active metabolite of vitamin D3, (1,25(OH)2D3; VD3). While both induce differentiation, differences in their mechanisms of action, as well as how the end states of the differentiation process differ, are not well understood. Therefore, this study used computational approaches to compare the effects of PMA and VD3 on the differentiation of monocytes into macrophages, using the promonocytic THP-1 cell line. Through the use of RNA-sequencing, gene expression was quantified in differentiated and undifferentiated THP-1 cells, treated with both PMA and VD3. Differential gene expression analysis was performed to determine genes that were differentially expressed as a result of either treatment relative to the untreated cells. This was followed by over-representation analysis to determine the pathways and processes in which the differentially expressed genes (DEGs) were involved. PMA treatment (3 989 DEGs) resulted in more changes in expression relative to VD3 treatment, where only 384 genes were found to be differentially expressed in response to treatment with VD3. Only TFE3, KIT and TRIB1 were observed to be crucial to the process of differentiation, irrespective of treatment. Apart from this, the treatments were observed to largely involve different biological pathways, resulting in cells that were phenotypically distinct from each other at the transcriptional level. This included changes observed in the expression of genes encoding transcription factors known to be involved in the differentiation process, such as CEBPA, GATA2, IRF8 and PU.1, as well as those encoding surface markers representative of monocytes and macrophages, such as CD14, CD64 and CD11b. The expression patterns observed here indicate that, at least at the concentrations and time points included in this study, PMA and VD3 induce macrophage-like cells that are at different stages of differentiation and are not comparable to either each other or primary macrophages. Furthermore, key differences observed in the expression of genes encoding pathogen recognition receptors and cytokines suggest that which differentiation inducing agent is used may have important implications for these cells’ capacity to recognise pathogens and produce cytokines. The findings of this study therefore emphasise that it is crucial to carefully consider the choice of differentiation-inducing agent when using THP-1 cells as an experimental system for studying monocyte-to-macrophage differentiation.
Differential Gene Expression Analysis of PMA Treated Pro-monocytic Cell Lines
(University of the Witwatersrand, Johannesburg, 2023) Kama, Asavela Olona; Meyer, Vanessa; Gentle, Nikki
HL-60, THP-1, and U937 are model cell lines that can undergo myeloid differentiation in vitro, allowing the study of myeloid cell function in drug metabolism, cytotoxicity, and the aetiology of infections. However, the differentiated end-state of these cells is not well characterised. Moreover, cell line-specific differences in the level of gene expression may confound results obtained from such studies. The aim of this study was thus to compare changes in gene expression between HL-60, THP-1, and U937 cells in response to the differentiation agent, phorbol 12-myristate 13-acetate (PMA), 48 hours after treatment. Gene expression profiles were compared across all three cell lines prior to and post-PMA treatment. Differential gene expression analysis between treated and untreated cells was performed using DESeq2 (v 4.2). Gene over-representation analysis was performed using cluster Profiler (v 4.0). HL-60, THP-1, and U937 cells had similar expression profiles prior to PMA treatment, but different sets of genes were significantly differentially expressed in these cell lines 48 h after treatment with PMA. A total of 475 genes were consistently differentially expressed across all cell lines. These genes were found to be involved in phagosome formation and cell cycle transition. HL-60, THP-1, and U937 cells had 944, 1231, and 624 uniquely differentially expressed genes, respectively. These genes were predominantly involved in energy metabolism and pathogen recognition. Overall, THP-1 cells showed greater potential to detect viruses, while U937 cells showed greater potential to detect bacteria. From this, it can be concluded that while all three cell lines did indeed undergo myeloid differentiation, the macrophage-like cell state produced in each case differed between cell lines.
Effects of acidification on the survival of pathogens in reconstituted infant formula
(University of the Witwatersrand, Johannesburg, 2023-10) Nemakonde, Mufunwa; De Maayer, Pieter
Infants are at a high risk of developing food-borne illnesses due to the consumption of powdered infant formulas (PIFs) contaminated by pathogenic microorganisms. These pathogens may be introduced during PIF production, transportation or during preparation and storage due to poor hygiene practices. Therefore, it is important to improve the microbiological safety of PIFs to reduce illness. Manufacturers have added further measures to ensure continued control of pathogenic microorganisms during the reconstitution and consumption, including the addition of prebiotics and probiotics, as well as acidification of the PIFs, which negatively affect the growth of pathogens. Different organic acids have been biologically or chemically added to commercial PIFs, however, little is known about the efficacy of the different acids in controlling pathogen growth post-reconstitution. The present study aimed to evaluate and compare the effects of two acids, namely citrate and lactate, integrated in commercial PIFs from two manufacturers on pathogen growth within the products. In Chapter 1, we present a review of the current literature pertaining to PIF production and consumption, its microbiological safety and key problem pathogens, as well as measures to control these pathogens (with specific emphasis on PIF acidification). In Chapter 2, the methodology of the present study is discussed. The effects of lactate and citrate incorporated in commercial PIFs against eight key PIF pathogens was evaluated using plate-based assays. Furthermore, the efficacy of PIF acidification at different storage temperatures and over prolonged storage periods was determined. Finally, to validate the effect of acidity on controlling pathogen growth, a spectrophotometric approach was used. Chapter 3 presents and discusses the key findings of the study. PIF acidification was found to negatively affect the growth of pathogens in comparison to the non-acidified infant formula. Acidification and storage at suboptimal temperatures resulted in little to no microbial growth. Lactate acidification of PIFs demonstrated greater inhibitory effects against most pathogens compared to citrate acidification. Further, lactate was found to have a bactericidal effect on the growth of some pathogens. Similarly, Luria Broth acidification resulted in the reduction of microbial growth. This validated that the acids and not other constituents of the infant formula were responsible for the inhibitory effect. Finally, Chapter 4 provides a summary of the key findings as well as recommendations and guidance on future studies which could be undertaken to enhance the effective control of pathogens in powdered infant formulas, thereby ensuring a safe and nutritious food source for infants.
Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies
(University of the Witwatersrand, Johannesburg, 2023-07) Van Deventer, Ruan; Achilonu, Ikechukwu Anthony
NNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.
Establishing and characterizing organoid cultures from colon tissue of South African individuals
(University of the Witwatersrand, Johannesburg, 2024) Du Plessis, Thea-Leonie; Kaur, Mandeep
Colorectal cancer (CRC) has been poorly studied in South Africa, with limited studies on disease progression and development. Studies that have investigated CRC in South Africa have indicated that there is racial disparity between different racial groups that may be attributed to alternative developmental pathways, differences in genetic compositions or CRC initiators that result in these different clinical presentations. Furthermore, the lack of population-based studies substantiates the need for more intensive CRC research. A particular model used to study cancer in general is the use of two-dimensional (2D) cell cultures, which have provided novel insight into many cancers and their development processes. However, these models lack the complex biology observed in vivo. One such model that is gaining research interest is the use of three-dimensional (3D) organoid cultures. Organoids are derived from stem cells and are able to self-organize and mimic the corresponding organ from which they were derived. Research has indicated that organoids are able to maintain cell-type heterogeneity as well as gene expression levels that resemble the organ of origin. Therefore, this project aimed at standardizing a protocol to establish and characterise colorectal organoid cultures from South African patient-derived tissues. Patient samples were obtained from individual patients with informed consent and were processed to generate organoids. The morphology of the organoids was monitored across several days and across passages. Once the organoids had reached maturity and were at passage 2, characterization was performed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence which indicated that the genetic composition and spatial localization of cell types of interest in non-cancerous tissue was recapitulated in the organoids. Based on these observations, it is proposed that organoids could be a promising model to investigate CRC disease development and progression and potentially search for novel therapeutics. This project has established the protocols for growing and characterizing organoids from African samples and provides baseline data, and outlines the complexities and issues involved in growing organoid cultures for the future studies
Expression and Methylation of Peroxidasin in Breast Cancer Cell Lines
(University of the Witwatersrand, Johannesburg, 2023-07) Falkov, Jemma Lilian; Mavri-Damelin, Demetra
Peroxidasin (PXDN) is a haem-containing extracellular matrix peroxidase protein which forms hypohalous acids in the presence of hydrogen peroxide (H2O2). The predominant role of PXDN is that of a collagen IV crosslinker within the basement membrane. Increased collagen IV deposition has been linked to tissue invasion and metastasis in breast cancer and PXDN has also been shown to assist in the process of epithelial-mesenchymal transition (EMT) in cancer. Various cancer types display dysregulated levels of PXDN expression including breast cancer and this dysregulation has been associated with poor prognosis. This study aimed to investigate whether DNA methylation of the PXDN promoter may be a mechanism through which changes in PXDN expression observed in breast cancer are regulated. Non-invasive MCF-7 and invasive MDA-MB-231 cells were used as models for luminal A and triple negative breast cancer (TNBC) respectively. The HEK-293 cell line was used as a non-cancerous control cell line. DNA methylation levels of the PXDN promoter and PXDN protein expression was investigated in these cell lines through the methods of methylation sensitive PCR (MS PCR) and immunofluorescence microscopy. Relative levels of PXDN expression were determined through immunofluorescence microscopy. Corrected total cell fluorescence (CTCF) analysis of these images revealed the highest PXDN levels to be found within the invasive MDA-MB-231 cell line, which was double that of the MCF-7 cell line. All cell lines were treated with 10 nM β-Oestradiol, which caused an increase in PXDN expression within the MCF-7 and HEK-293 cell lines and a decrease in expression within the MDA-MB-231 cell line to half its untreated value. PXDN was found to be localised in the ECM in all three cell lines. To elucidate the role of DNA methylation, methylation sensitive PCR (MS PCR) was performed on all three cell lines, with four primer pairs spanning a region of 1305 base pairs (bp) within the PXDN promoter. A region of differential methylation was found between the MDA-MB-231 and HEK-293 cell lines between 524 bp and 53 bp upstream of the transcription start site (TSS). This region was unmethylated within the MDA-MB-231 cell line and methylated within the HEK-293 cell line, which correlates with expression differences between these two cell lines and suggests this region could be of regulatory significance. The four primer pairs designed to amplify the PXDN promoter were unable to amplify this region within the MCF-7 cell line. A heterochromatic DNA conformation or a point mutation increasing CpG content creating a thermodynamically ultra-fastened (TUF) region could be the explanation behind this phenomenon, however further research is required to elucidate the mechanism responsible. In conclusion, PXDN shows higher expression in TNBC cells than in luminal A subtype cells. The oestrogen receptor is involved in regulating PXDN expression, however, different mechanisms seem to be at play between the two cell lines. The contribution of CpG methylation to this change in PXDN expression remains unknown, as does the nature of the interaction between the oestrogen receptors and the gene. Further research is required to clarify the mechanisms involved.
Insights into the Physiochemical Properties of the Interaction between the L38↑N↑L+4 HIV-1 Hinge Mutant Subtype C Protease and a Related Gag Cleavage Site
(University of the Witwatersrand, Johannesburg, 2023-09) Adams, Taryn Racheal; Sayed, Yasien
Frequent mutations in HIV protein drug targets such as the protease (PR), have led to a rise in resistance to clinically available treatments and inquiries into the associated biochemical mechanisms. In this study, the L38↑N↑L+4 PR (mutant) and related gag, were isolated from a PR inhibitor naïve child for further study. Wild-type and mutant PRs were successfully overexpressed and purified using ion exchange chromatography. Intrinsic fluorescence studies probing tryptophan residues located at the hinge revealed variations in tertiary structure. This coincided with significant differences in refolding efficiency (mutant PR recovery ~ 10% and wild-type PR recovery ~ 34%). Furthermore, differences in catalytic efficiency (mutant-specific activity ~ 7.4 µmol.min-1.mg-1, mutant kcat ~ 2.71 sec-1; wild-type specific activity ~ 31.6 µmol.min-1.mg-1, kcat ~ 11.60 sec-1). Thermal shift assays revealed reduced mutant PR structural stability (Tm ~ 67 ℃) compared to the wild-type PR (Tm ~ 64 ℃). Furthermore, reduced stability of inhibitor-mutant PR complexes (Tm ~ 73.5 ℃ Acetyl pepstatin (AP), 90 ℃ Darunavir (DRV), and 88 ℃ Saquinavir (SQV)) compared to complexes involving the wild-type PR (Tm ~ 70 ℃ (AP), 82 ℃ (DRV), and 82 ℃ (SQV)). Overall, the L38↑N↑L+4 PR mutations were found to influence the tertiary structure of the hinge, gag processing, and PI stability within the mutant PR active site. Computational docking studies highlighted the potential role of a gag single nucleotide polymorphism (SNP), located at the 4th amino acid (P4) position of the peptide-2-nucleocapsid (p2/NC) gag cleavage site, for future studies as a compensatory mutation aiding PR polymorphisms. Further studies should focus on the gag-PR functional pair to build a more accurate understanding of HIV drug resistance mechanisms.
Investigating the susceptibility of Enterobacter xiangfangensis Pb204 to various silver(I) phosphine compounds and identifying the molecular basis of resistance though comparative genomics
(University of the Witwatersrand, Johannesburg, 2023-10) Ntuli, Phindile Lindiwe; Engelbrecht, Zelinda; Cronjé, Marianne J.
In developing countries, the endemic of nosocomial infections is notably high, particularly in the ICU settings and neonatal infections, where it accounts for a prevalence of 15.5% with an increased mortality rate. ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) pathogens are highly virulent and resistant microbes that predominantly causes nosocomial infections in clinical settings. These pathogens adhere to the surface of hospital equipment and form biofilms, thereby increasing the susceptibility of patients to acquiring such infections during extended hospital stays. Recent studies have shown that Enterobacter xiangfangensis is the main clinical culprit in the Enterobacter cloacae complex, thus making it the main therapeutic target among the ESKAPE pathogens. This study evaluated the antimicrobial properties of various silver(I) phosphine compounds on E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T. The minimum inhibitory concentration (MIC) assay was done, though the microdilution technique coupled with iodonitrotetrazolium chloride (INT) dye. The antibiofilm activity of the silver(I) phosphine compounds on biofilm formation and degradation were evaluated using the crystal violet assay, and the metabolic activity of cells in biofilms was assessed using the XTT assay. Additionally, the molecular mechanism of silver resistance was investigated by measuring gene expression of E. xiangfangensis Pb204 cells when treated with the silver(I) phosphine compound UJ1, silver nitrate and silver sulfadiazine using RT-qPCR. The MIC of the silver(I) phosphine compounds was 0.125 mg/ml for both E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T. Moreover, the compound UJ1 showed a bactericidal effect against E. xiangfangensis LMG 27195𝑇. Furthermore, the silver(I) phosphine compounds have antibiofilm activity since they inhibit biofilm formation, disrupt matured biofilms, and reduce metabolic activity in E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T biofilms. The resistance in E. xiangfangensis Pb204 strain is due to the upregulation of the silver resistance pathway encoded on the integrative and conjugative element (ICE) element. Core and unique protein coding genes between E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T were identified though comparative genomics. Functional annotation of core and unique protein revealed that E. xiangfangensis Pb204 evolved to gain genetic material coding for inorganic ion transport and carbohydrate transport metabolism. Knowledge of these can be used in future studies in the utilization of silver metals as antimicrobial agents.
Probing the Protein-Protein Interactions of the FOXP1, FOXP2 and FOXP3 Forkhead Domains
(University of the Witwatersrand, Johannesburg, 2023-07) Mhlongo, Paulina; Fanucchi, Sylvia
The FOXP proteins are classified amongst the forkhead box superfamily of transcription factors due to their highly conserved forkhead winged-helix domain (FHD). It is through this domain that FOX transcription factors are able to bind DNA in order to perform crucial roles in the regulation of gene transcription from development through adulthood. The FHD of FOXP1, FOXP2 and FOXP3 is remarkably unique in its ability to establish domain-swapped dimerization, postulated to drive interchromosomal interactions to regulate the transcription of distal genetic material. The FOXP1 and FOXP2 proteins are co-expressed and have also been demonstrated to directly interact in vivo and in vitro via a domain upstream of the FHD. Similarly, the FOXP1 and FOXP3 proteins have been established to form direct heterotypic interactions to function in regulatory T-cells (Treg). Provided that the FOXP FHD has the exceptional capability for dimerization, the study of the heterodimerization of the FHDs of these FOXP transcription factors may provide insight into the role of FHD-dimerization and its importance in the physiological roles of these proteins. Therefore, the aim of this work was to investigate whether a FOXP FHD heterodimerization event can occur between the FOXP1 and FOXP2 FHDs as well as between the FOXP1 and FOXP3 FHDs. The three FHD proteins were expressed and isolated for downstream interaction studies. The activity of the purified FHDs was studied using basic electrophoretic mobility shift assay. Their secondary and tertiary structures were characterized with circular dichroism and intrinsic fluorescence spectroscopies. Concentration-dependent size exclusion chromatography was employed to study their propensity for dimerization and fluorescence anisotropy was used to investigate both the homo and heterodimerization of the FHDs. It was revealed that FOXP3 FHD showed the highest propensity to homodimerize, whilst FOXP2 FHD showed the weakest propensity. Despite this, the homodimers of FOXP1 FHD appear to be less stable than that of the FOXP2 FHD. The heterodimerization studies suggest that FOXP1 FHD has preference in forming heterotypic associations with FOXP3 FHD rather than FOXP2 FHD.
Structure-activity relationship between Klebsiella pneumoniae β- Lactamase CTX-M-15 and selected β-lactam antibiotics: Evaluating the binding site promiscuity
(University of the Witwatersrand, Johannesburg, 2023-08) Esau, Veruschka Chloe-Zaan; Sayed, Yasien; Achilonu, Ikechukwu Anthony
Background: Nosocomial infections have become a major concern in Sub-Saharan Africa. Bacteria predominantly cause these infections due to resistance development attributed to lack of novel therapeutics. These resistant bacteria are classed as ESKAPE pathogens that gained resistance to most known antimicrobials; one such is the Gram-negative Klebsiella pneumoniae. K. pneumoniae developed resistant strategies against most known β-lactam antibiotics through the development of the enzyme, β-lactamase. Beta-lactamases hydrolyse the β-lactam ring of β-lactam antibiotics rendering them ineffective towards K. pneumoniae. This research aimed to investigate the structural and functional characteristics of K. pneumoniae β-lactamase and asses the conformational stability with penicillin and cefoperazone (CPZ). Methods: Overexpression of recombinant wild-type and mutant-(S70A) K. pneumoniae β-lactamase in E. coli T7 cells using a pET-28a vector and protein purification using Immobilised Metal Affinity Chromatography (IMAC), and enzyme activity assessed with nitrocefin. Secondary, tertiary, and quaternary structure studies were conducted with Far-UV CD, tryptophan fluorescence, ANS fluorescence and size exclusion HPLC respectively. The thermal stability and binding mechanisms were assessed with thermal shift assay and isothermal titration calorimetry (ITC). Results: The wild-type and mutant-(S70A) K. pneumoniae β-lactamase was successfully expressed and purified. S70A-KpBlac-1 showed no activity towards nitrocefin, and WT-KpBlac-1 was highly activity towards nitrocefin. The native structures were determined to be alpha-helical, but alpha-helical content is lost upon penicillin and CPZ binding to unorder and β-stranded conformations. The binding site was determined to be solvent exposed with one hydrophobic active site. The proteins were monomeric. CPZ induce thermal stability on S70A-KpBlac-1, whereas as penicillin binding had no effect on the thermal stability. Penicillin binding to S70A-KpBlac-1 was endothermic and the protein had low binding affinity for penicillin. CPZ binding was exothermic, and the protein had higher binding affinity for the substrate. Conclusion: Potential novel inhibitor design should be focused on CPZ. Through substrate-based drug discovery, potential drugs should confer a similar shape, size, or stereochemistry as CPZ. This would change the conformation of the protein, be tightly bound to the active site, and lower inhibitor concentration would be required. Therefore, these findings contribute, and provide insights on potential novel inhibitors against highly antibiotic resistant bacteria.

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