3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The design and synthesis of A54556 acyldepsipeptide derivatives and nanomaterials targeting the mycobacterium tuberculosis caseinolytic protease(2024) Cobongela, Sinazo Zezethu ZongeziweAntibiotics have saved many lives since their discovery in the 1920s, and continue as a first line of defence against microbial infections to date. Unfortunately, their misuse gave rise to the multi-drug resistant (MDR) bacteria that require intensive therapy. Mycobacterium tuberculosis (M. tb) is one of the top pathogens rapidly gaining resistance to current treatment regimens. Over 480 000 cases of MDR tuberculosis (TB) occur every year globally, 9% are caused by the extensively drug-resistant (XDR) M. tb strains. The treatment of MDR/XDR-TB is long, toxic and expensive, and the success rate is largely unsatisfactory. Hence, there is an urgent need for new and effective anti-TB drugs. Over the years, peptide based therapeutics have been used and were successful in treatment of various diseases such as TB, cancer, cardiovascular diseases, diabetes and others. Due to the instability, poor solubility and toxicity of natural antimicrobial peptides (AMPs), great attention is now focussed towards synthetic analogues of the AMPs to improve their pharmacokinetics. One of the new antibacterial targets that have gained traction over the years is the proteolytic regulating protease called Caseinolytic protease (ClpP). ClpP degrades misfolded and aggregated proteins and peptides that have potential to induce toxicity within the bacterial cell. ClpP can be indirectly targeted by dysregulating ClpP-ATPases such as ClpC1 and ClpX, which are responsible for activation of the ClpP proteolytic activity. Acyldepsipeptides (ADEPs) function by mimicking the binding of ClpP-ATPases to ClpP, thereby inducing nonselective proteolysis, which ultimately leads to bacterial death. Due to the essential role played by ClpP in the biology of the M. tb, thus ADEPs are regarded as potential antibiotics with a novel mechanism to combat the bacterial resistance problem. However, ADEPs also have limitations such as poor membrane permeability and high cytotoxicity. Therefore, the aim of this study was to design and synthesise biocompatible ADEP1 analogues as potent antibacterial agents, and to improve their membrane permeability and efficacy by conjugating the analogues to Silver/Indium/Sulphide (AgInS2) quantum dots (QDs) as nano-carriers. Twenty cyclic ADEP1 analogues were designed and synthesised using the solid phase peptide synthesis (SPPS) strategy. Four of the ADEP1 analogues were conjugated to the AgInS2 QDs, and characterised with UV-Vis, Spectro Xepos05 energy-dispersive X-ray fluorescence, highresolution transmission electron microscope, and spectrofluorophotometer. The antibacterial activity of the ADEP1 analogues and AgInS2 QDs-ADEP1 analogue conjugates was tested against two Gram-negative bacteria, Escherichia coli (E. coli) and Pseudomonas aeruginosa vi (P. aeruginosa), and three Gram-positive bacteria, Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus), including Methicillin-resistant Staphylococcus aureus (MRSA). The biocompatibility and cytotoxicity effects of the ADEP1 analogues and AgInS2 QDs-ADEP1 analogue conjugates was evaluated on HEK-293 and Caco-2 cells using the MTS viability assay. The effect of the ADEP1 analogues on the peptidase activity of the M. tb ClpP1P2 was investigated. The aqueous solubility and membrane permeability of the ADEP1 analogues was investigated using the MultiScreen aqueous solubility assay and the Parallel Artificial Membrane Permeability Assay (PAMPA), respectively. Four of the ADEP1 analogues were successfully synthesized by SPPS with >96% purity and >36% yield. Both the ADEP1 analogues and AgInS2 QDs-ADEP1 analogue conjugates showed potency against Gram-positive and Gram-negative bacteria, however, the efficacy of the ADEP1 analogues was significantly enhanced when conjugated to the AgInS2 QDs. In Gram-positive bacteria, the ADEP1 analogues exhibited a minimum inhibition concentration (MIC) ranging from 63-125 µM while the AgInS2 QDs-ADEP1 analogue conjugates had lower MIC values (9.38-18.75 µM). The MIC values of ADEP1 analogues against Gram-negative bacteria were between 125 and 500 µM while the AgInS2 QDs-ADEP1 analogue conjugates had MICs between 2.34 and 37.5 µM. Both the unconjugated peptides and conjugates showed minimum bactericidal concentrations (MBC) twice their MIC. In addition, the ADEP1 analogues and AgInS2 QDs-ADEP1 proved to be biocompatible as they were non-toxic to the cells. The ADEP1 analogues further showed moderate aqueous solubility and gastrointestinal membrane permeability. Furthermore, the effect of the ADEP1 analogues on the peptidase activity of the M. tb ClpP1P2 protease was investigated. All the analogues enhanced M. tb ClpP1P2 activity. Of the four ADEP1 analogues that were tested, SC008 and SC005 showed activation percentages that were 32% and 14% higher than benzoyl-leucyl-leucine (Bz-LL), a known ClpP1P2 activator, respectively. These results suggested the potential use of these novel ADEP1 analogues as leads in the development of broad spectrum anti-bacterial agents. The ADEP1 analogues also demonstrated potential anti-mycobacterium activity by activating M.tb ClpP1P2. The mode of action followed by the ADEP1 analogues is an interesting finding that can be explored to develop new and effective antibacterial drugs with potential to eradicate drug resistance. Therefore, future studies warrants investigation of the anti-TB effects and molecular mechanism of the ADEP1 analogues and conjugates in in vitro and in vivo models of M.tb.Item Cross-resistance among rifamycins in mycobacterium tuberculosis clinical isolates(2019) Mojapelo, Richard MarediIntroduction: Rifamycins (RFMs) are a group of antimycobacterial drugs that belong to the large family of ansamycin. RFMs inhibit Mycobacterial growth by blocking the RNA polymerase subunit B (rpoB). High level cross-resistance among RFMs in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates is commonly inferred. However, previous studies reported that the minimum inhibitory concentrations (MICs) of rifabutin (RFB) among rifampicin (RIF)-resistant M. tuberculosis carrying rpoB mutations varies depending on the mutation position. Objective: -To determine the proportion of cross-resistance among rifamycins and to assess the use of the GenoType MTBDRplus Version 2.0 assay in predicting differential susceptibility to rifamycins in M. tuberculosis isolates. Method: -A total of 300 unique baseline isolates which were collected between June 2015-April 2016 for routine laboratory based surveillance of RIF drug resistance in selected districts of South Africa were included. Drug susceptibility testing (DST) for RIF (1.0 μg/ml), RFB (0.5 μg/ml) and rifapentine (RFP) (0.5 μg/ml) was performed by the MGIT 960 system using World Health Organisation (WHO) recommended critical concentration (c.c). The MycoTB plate was used to determine MICs for RIF and RFB. To determine rpoB mutations, all the isolates were tested by Genotype MTBDRplus version 2.0 assay method and undefined isolates were sent for Sanger sequencing. Results: -The proportion of cross resistance among RFMs were: across all three (216/300;72%), between RIF and RFB (217/300;72%) and RIF and RFP (292/300;98%). The S531L mutation was the mostly associated with cross resistance to all RFMs (144/153;94%), while the D516V mutation was associated with differential susceptibility to RFB (50/52;96%). Conclusion: -The results show high levels of cross resistance across all rifamycins, however 28% of MDR/XDR-TB cases could potentially benefit from RFB as a substitute drug to the failing RIF. These findings provided additional evidence of the strong association of specific rpoB mutations with the development of RFMs cross and differential susceptibility. The use of LPA and rpoB mutations specifically S531L and D516V can be beneficial in rapidly differentiating phenotypic differential susceptibility to RFB according to this study.Item Application of Computer Vision and Machine Learning for classification of mycobacterium Tuberculosis (TB) cultures to assist disease diagnostics(2019-10) Venkataraman, KarthikeyanDiagnosis of Tuberculosis (TB) by the analysis of sputum samples is a manual activity that requires the sputum culture samples to be analysed after a gestation period between 6 to 12 weeks following the collection of the sample from the patient. The current method of TB diagnosis at the National Heath Laboratory Service (NHLS) is a manual process that involves photographing the culture samples by the laboratory technician using a camera phone, among other steps. It is proposed to use these photographs to automate the process of diagnosis by using Computer Vision and Machine Learning technologies to detect the presence of Tuberculosis bacteria. A photograph of a culture sample or plate, consists of 48 unique circular regions or wells, each of which is classified by the machine learning program as Positive, Negative, Contaminated or Condensation (indicating presence of water bubbles). This classification of each well is done using a combination of Image Processing, Computer Vision and Machine Learning (ML) techniques. Two ML programs, in particular, were used for this task - Gaussian Mixture Models (GMM) and Random Forest (RF). At the time of writing of this report, a classification accuracy of 77.6% using 25 training images was obtained. The results from each ML technique are compared and approaches to further improve the classsification accuracy are discussed. A data flow and machine learning pipleline was developed in Python 3 and the code has been opensourced on GitHub at https://github.com/karthik111/TB-culture-classification-ML allowing future work to be undertaken. The Image Processing and Computer Vision techniques implemented here are agnostic of the downstream ML technique allowing other ML approaches to be explored. This work establishes the feasibility of an automated approach for classification of sputum samples to aid TB diagnosis and describes approaches that if taken, can further improve the classification accuracy.Item The design and synthesis of antituberculosis peptidomimetics focusing on lassomycin derivatives(2019) Ngqinayo, NtombizaneleTuberculosis (Tb) is a disease ranked among the top ten causes of death worldwide and is responsible for infecting around 10 million people each year. Tb is caused by the Mycobacterium Tuberculosis (M. tb) bacterial pathogen. The mycobacterium has become resistant towards currently approved drugs which mostly target the cell wall and this has led to the development of the multidrug resistant (MDR) and extremely drug resistant (XDR) M. tb strains. The resistant strains are difficult to treat and require longer treatment duration with the use of combinatory drugs that result in a number of serious side effects. These limitations have led to the search for novel anti-Tb agents and the discovery of lassomycin, an antimicrobial peptide (AMP) that utilizes a different mode of action. The peptide targets the caseinolytic protease of M. tb which is essential for cell survival and causes uncontrolled protein unfolding which results in cell death. Lassomycin is a 16amino acid long basic peptide isolated from a soil bacteria, Lentzea kentuckyensis sp. that been found to be highly selective and potent towards M. tb without affecting mammalian cells.2 The objectives of this project are (i) to modify lassomycin into drug-like derivatives by incorporating N-methylated amino acids to make the peptide more stable against enzymatic degradation; (ii) to shorten the synthetic route by replacing the lactam bridge with a disulfide bridge; (iii) to replace the arginine amino acids in the peptide sequence (difficult to couple) with lysine amino acids to investigate the role of arginine in the binding of the peptide to the acidic region of the caseinolytic enzyme; and (iv) to make the peptide more cationic to improve selectivity for the negatively charged bacterial membrane by adding lysine residues. Peptides were synthesized via the fmoc solid phase peptide synthesis strategy; purified using a semi-preparative High Performance Liquid Chromatogram (prep-HPLC) and analyzed using High Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The bactericidal activity of selected lassomycin derivatives against M. tb was determined using the Alarmar blue assay and one of the derivatives showed a bactericidal effect at a concentration of 9.87 µg/ml which is comparable to that of ethambutol. The derivatives were also found to be selective for pathogens that share a similar protease to that of the M. tb such as Bacillus Subtilis (B. subtilis) and inactive against other pathogens that do not contain the protease. The 3-dimensional structure of the active derivatives will be determined in the future using NMR spectroscopy.Item Evaluation of new diagnostic methodology for the detection of second-line drug resistance in Mycobacterium tubercolosis clinical isolates(2018) Gardee, YasminThe World Health Organization (WHO) Global Report, 2017, confirms that tuberculosis (TB) is the primary cause of death globally caused by an infectious agent. Drug-resistant TB (DR-TB) is reported by the WHO to affect at least 600 000 people globally and is the primary obstacle in the fight against the elimination of TB disease. The prompt identification and treatment of DR-TB is essential. Conventional culturebased diagnostic tests for TB require substantial laboratory capacity and result availability can take up to 3 - 4 months. The implementation of rapid molecular diagnostic tests such as line probe assays (LPA) and the Xpert MTB/RIF® (Cepheid, USA) have led to an improvement in reporting turnaround times. GenoType® MTBDRsl VER 2.0 LPA is designed for molecular detection of second-line drug resistance-conferring mutations in genes encoding resistance to fluoroquinolones (FLQ) (gyrA and gyrB) and second-line injectable drugs (SLID) (rrs and eis). This study evaluated the diagnostic performance of the Genotype® MTBDRsl VER 2.0 compared to phenotypic drug susceptibility testing (DST) as the gold standard, on clinical samples and Mycobacterium tuberculosis complex clinical isolates from South Africa. The performance indices (sensitivity and specificity estimates) for the assay when tested on clinical samples were as follows respectively: FLQ 77.2% (95% CI, 67.2% - 85.3%); 85.4% (95% CI, 79.0% - 90.5%) and SLID 81.9% (95% CI, 75.5% - 87.2%), 91.5% (95% CI, 86.6% - 95.5%). For clinical isolates the performance indices were as follows respectively: FLQ 100% (95% CI, 95.8% - 100%); 98.9% (95% CI, 96.1% - 99.9%) and SLID 89.2% (95% CI, 79.1% - 95.6%); 98.5% (95% CI, 95.7% - 99.7). The incorporation of the assay into the TB diagnostic algorithm will improve South Africa’s TB Control Program in prompt identification and appropriate treatment of drugresistant TB cases.Item Comparison of vaccine and wild- type measles virus strains effect on polymorphonuclear leukocyte function : an in vitro analysis.(2001-08-31) Phungwayo., Mokete. Amos. NehemiaMeasles virus (MV)-infected persons have been found to be at a greatly increased risk of developing respiratory infections caused mainly by Staphylococcus aureus and Mycobacterium tuberculosis. Secondary microbial infections have been implicated as important causes of morbidity and mortality in patients with measles. Polymorphonuclear leukocytes (PMNL), the first cells of the immune system to migrate to the site of microbial invasion, are the principal cells involved in the immune response to invasion of the host by microbes, particularly bacteria and fungi.Item Phenotypic and genotypic characterisation of mycobacterium tuberculosi strains in relation to the transmission of tuberculosis in South African mines(2000) Muthivhi., Tshilidzi, Neleus.The prevalence of tuberculosis in South African miners is substantially higher than that of in the general population. Through exposure to dust which leads to different degrees of silicosis, and by working in enclosed spaces where coughed out bacilli can survive in droplet nuclei and be inhaled by other workers, miners are especially prone to to become infected with M. tuberculosis and develop the disease. It is not only the working conditions which promote transmission of M. tuberculosis, but the living conditions as well. Most miners live and sleep in rooms shared by up to eight other men, which increases the opportunity for transmission, leading to both primary and reinfection tuberculosis.Item The role of mononuclear cells in tuberculosis(1992-06-03) Sussman, GarthSonicates derived from Mycobacterium tuberculosis suppressed lymphocytes proliferation. Pulsing of monocytes with mycobacterial sonicates resulted in the release of high molecular weight lipids. Both these lipids and those prepared by column fractionation of mycobacterial sonicates suppressed lymphocyte blastogenesis.This effect was due to the activation and not the proliferation of CD8+ lymphocytes by the lipid containing mycobacterial fractions of Mr>200kDa that could be obtained in vitro by column fractions.Item Crystal and co-crystal Engineering of Isoniazid, 4-Aminoantipyrine, benzophenone and their derivatives(2018) Smith, Mark GordonIsoniazid is the primary drug used for the treatment of tuberculosis. Covalent modifications to the amine group of isoniazid are currently under investigation and such modifications have been shown to render the drug effective against multiple-drug-resistant strains of Mycobacterium tuberculosis. In this thesis, crystal and co-crystal engineering was applied to synthesize crystals and co-crystals of covalently modified isoniazid. Isoniazid was modified by bulky modifiers, namely benzophenone and benzophenone derivatives, and crystallized in the absence and presence of co-formers. A series of co-crystals of modified isoniazid was prepared and their supramolecular structures were studied. All co-crystals were synthesized with salicylic acid and control over the dimensionality of packing was achieved. It was found that the benzophenone modifiers sterically masked the amide functional groups from participating in any intermolecular interactions. This steric masking is predictable and consistent and provides a general method whereby an amide functional group can be sterically blocked from intermolecular interactions. The effects of reaction and crystallization conditions on the supramolecular structure of isoniazid are also reported. Several pairs of co-crystals of modified isoniazid were synthesized under different crystallization conditions. Change in reflux time of the reagents resulted in stoichiometric variation in the resulting co-crystals. The addition of excess reagents as an additive resulted in polymorphism. Addition or absence of an acid catalyst either promoted or prevented solvate formation. The addition of either one or two methyl groups to the benzophenone aromatic rings did not substantially alter the packing patterns of the crystal structures. As the presence of co-formers may either enhance or inhibit the bioavailability of pharmaceutical drugs, the covalent modification of isoniazid was repeated in the absence of co-formers. The covalent modification without co-formers presented several synthetic difficulties and use of new catalysts and the development of new synthetic strategies such as refluxing in high-pressure glass vials were developed. A full structural study on the covalently modified isoniazid crystals is reported. Side reactions of benzophenone were observed during the crystallization of isoniazid derivatives. These resulted in the formation of benzophenone azine crystals. The origin and mechanism of the formation of these benzophenone azine crystals were determined to occur via a two-step photodimerization process. The benzophenone azine crystals were found to be held together through weak interactions and a structural study of the packing of these crystals was reported. 4-Aminoantipyrine is a pharmaceutical drug previously used as an antipyretic and is considered a prophylactic against oxidative stress. Two of its derivatives, aminoantipyrine and 4-(N,N-dimethyl)-aminoantipyrine, have been used as analgesic and anti-inflammatory drugs since the late 19th century. Their concurrent use with aspirin is particularly beneficial as they have been shown to prevent or attenuate the anti-platelet effects of aspirin. However, their use has been discontinued due to side effects and this discontinuation warrants further studies of methods to possibly increase the bioavailability and alter the physical properties of the drug, including via covalent modification and co-crystallization. To date, all reported attempts at co-crystallizing this drug have failed. In this study, co-crystal design strategies were developed to synthesize the first co-crystal of 4-aminoantipyrine, and the methods leading up to the successful supramolecular synthesis of 4-aminoantipyrine with co-formers are detailed. The synthesis of two novel salts of 4-aminoantipyrine and two co-crystals of covalently modified 4-aminoantipyrine are also reported. A series of crystals of covalently modified 4-aminoantipyrine were also synthesized and a full structural study of these crystals is presented.Item Characterization of mycobacterial peptidoglycan remodelling enzymes(2018) Mashilo, PoppyMycobacterium tuberculosis (TB), the causative agent of tuberculosis, is responsible for over one million deaths per annum, a substantive proportion of these due to multidrug resistant or extensively drug resistant strains. The available antibiotics are rapidly becoming ineffective due to the development of resistance mechanisms by the pathogen. Considering this, there is an urgent need for novel and highly effective new TB drugs, with novel modes of action. The peptidogycan (PG) layer in the cell wall has emerged as a rich area for drug discovery. It undergoes constant reconstruction by penicillin binding proteins (PBPs) and other enzymes to allow for cell growth and division, while preventing lysis. In this study, we characterize the function of two Low Molecular Mass PBPs, known as D,D-carboxypeptidases (D,D-CPases, MSMEG_6113 and MSMEG_2433) in Mycobacterium smegmatis, a model organism for TB research. Using a bacterial two hybrid system, we demonstrate that MSMEG_2433 interacts with PonA1 (A High Molecular Mass PBP involved in PG synthesis) and FtsH (An AAA family protease and a member of the divisome complex). We also demonstrate that MSMEG_6113 forms a complex with FtsI (A High Molecular mass PBP and also part of the divisome complex) and a cell division control protein, Cdc48. We demonstrate that the two D,D-CPases associate with their partnering proteins via their C-terminal transpeptidase domain. The importance of these identified interactions for cell division and growth was tested through deletion of partnering proteins from the mycobacterial genome, particularly FtsI and Cdc48. FtsI is essential for mycobacterial growth in vitro as demonstrated by the inability to recover mutants through allelic exchange by homologous recombination, while Cdc48 is dispensable for growth. We noted morphological and cell division defects in the Cdc48 deletion mutant strain. The absence of Cdc48 results in bulging, kinking and chaining phenotypes, in addition to misplacement of the FtsZ ring. Collectively, our observations describe the presence of novel PG hydrolysing protein complexes that may mediate essential steps in PG synthesis and bacterial proliferation. Targeting these complexes may provide an attractive avenue for the development of novel TB therapeutics.