3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Mycobacterium amidases: biological function and putative role in cell wall remodeling(2018) Sikhosana, NombekoMycobacterium tuberculosis, the causative agent of tuberculosis, uses macrophages to cross host mucosal barriers and initiate infection at sterile sites in the alveoli, where it establishes infection and evades immune detection. Despite significant effort, the mechanism through which mycobacteria replicate and persist within host tissues remains enigmatic. Bacterial replication requires substantive rearrangement of the cell surface and in this context, the bacterial cell wall and the enzymes that remodel the various polymers present in this structure are of particular interest. Within the cell wall, the bacterial peptidoglycan (PG) layer has been the subject of intense research, given the therapeutic benefit of targeting this macromolecule in various diseases. PG hydrolases specifically, N-acetylmuramyl-L-alanine amidases (amidases) in other bacterial species have been shown to contribute to essential cell division processes and pathogenesis. In this study, the role of mycobacterial amidases in cell growth and division was investigated to assess their function in Mycobacterium smegmatis, a model organism routinely used for tuberculosis research. For this, putative amidase-encoding genes ami3, ami4 and ami5 were individually deleted using homologous recombination. The resulting mutant strains were then phenotypically characterized by investigating nascent cell wall incorporation, cell elongation and division mechanisms. Deletion of ami3 resulted in severe cell division defects, in many cases these were associated with mislocalization of the cell elongation and division apparatus. In addition to this, cell wall permeability was severely affected, with the strain displaying increased susceptibility to cell wall targeting antibiotics. Deletion of ami4 resulted in the formation of twisted and morphologically atypical cells, some of which died after the first cell division event. In contrast, deletion of ami5 resulted in the formation of short cells, suggesting that this enzyme plays a role in cell growth/elongation. Collectively, these data describe novel and non-redundant roles for amidases in mycobacterial cell growth and division, thus validating them as potential new drug targets for tuberculosis diseaseItem Molecular basis of the interplay between the Nth and Nei DNA glycosylases of the base excision repair pathway in Mycobacterium smegmatis(2017) Rantsi, Tebogo ChristinaDuring infection, Mycobacterium tuberculosis encounters hostile conditions which result in the generation of host-derived reactive oxygen (ROS) and nitrogen species (RNS) as part of the immune response to control the infection. Exposure to these reactive radicals can lead to oxidative damage of DNA, which ultimately destabilises the genome and introduces mutations. However, M. tuberculosis is well equipped with a number of DNA repair pathways such as the base excision repair (BER) pathway, which plays a role in maintaining genome stability and survival of the pathogen. A number of DNA glycosylases are involved in the BER pathway, including formamidopyrimidine (Fpg), endonuclease VIII (Nei) and endonuclease III (Nth), which are the initial enzymes responsible for recognition and excision of damaged DNA bases. It was previously demonstrated that combinatorial deletion of nth and two nei homologues in Mycobacterium smegmatis resulted in reduced survival under oxidative stress conditions with a corresponding increase in mutation rates, suggestive of interplay between these enzymes. To understand the molecular basis of this interplay, the individual effects of the Nei homologues (NeiI and NeiII), together with Nth on survival and mutagenesis under oxidative stress conditions, expected to induce DNA damage, were investigated in the current study. Two mutants lacking nth and either neiI or neiII were generated by homologous recombination. These double deletion mutants together with the individual deletion mutants, the parental strain and the respective complemented strains were phenotypically characterized under oxidative stress conditions and assessed for increased mutagenesis as measured by rifampicin resistance. Defects in the BER system resulted in reduced survival under oxidative stress conditions. Deletion of nth combined with the neiII homologue led to reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin when compared to the deletion of nth combined with the neiI homologue. Collectively these data suggest that NeiII may play an important physiological role in BER in comparison to the NeiI homologue.Item Analysis of gamma-delta T cells in black South African patients with active tuberculosis(2014) Sedick, QanitaMycobacterium Tuberculosis is the leading cause of morbidity and mortality due to infectious diseases worldwide. South Africa has ~20% of the world’s HIV associated Tuberculosis and has the second largest reported numbers of multidrug resistant (MDR) Tuberculosis in the world. Given the complexity of the mycobacterium and its ability to evade the immune system, there is a need for dissecting the immunological response to Tuberculosis including innate like lymphocytes such as gamma-delta T cells. Gamma-delta T cells are of particular relevance as they react to phospho-proteins of mycobacteria. Gamma-delta T cells can be divided into two subsets. Gamma-delta T cells using the Vdelta2 (VD2) segment as the variable segment in their T cell receptor and gamma-delta T cells using an alternative variable segment (non VD2 T cells). We aimed to enumerate both subsets of gamma-delta T cells in the immunological response to Tuberculosis. We collected samples from three patient populations at the Charlotte Maxeke Johannesburg Academic Hospital for comparison: HIV positive patients with no evidence of Tuberculosis disease, HIV positive patients with active pulmonary Tuberculosis and a healthy control group. We used a nine colour flow cytometric panel to enumerate the frequency of gamma-delta T cells in these participant groups. We found that the VD2 T cell subset was reduced in the HIV positive group and the dual HIV positive TB positive group compared with healthy controls, which mirrored the loss of CD4 T cells in these patients. Conversely, the non VD2 subset of gamma-delta T cells showed a statistically significant increased frequency in HIV positive patients and dual HIV positive TB positive patients compared to healthy controls. The frequency of gamma-delta T cells, expressed as a percentage of total T cells, was significantly increased in HIV positive patients and not non- significantly increased in the HIV positive TB positive groups compared to healthy controls. This skewing of the gamma-delta T cell repertoire in HIV positive patients and HIV positive patients with active Tuberculosis may have specific immune implications. The mechanism of the loss of VD2 T cells in HIV and HIV associated Tuberculosis has not been elucidated. The loss of VD2 gamma-delta T cells in HIV and HIV associated Tuberculosis may underlie susceptibility to Tuberculosis disease.Item Construction and phenotypic characterization of Mycobacterium smegmatis mutants deficient in DNA glycosylases(2014-02-18) Moolla, NabielaThe causative pathogen of tuberculosis, Mycobacterium tuberculosis (Mtb), is equipped with several DNA repair mechanisms for continued survival within the host. One such mechanism is Base Excision Repair (BER) that repairs DNA damage caused by reactive oxygen and nitrogen species (ROS/RNS) generated by the host immune cells during infection. BER is dependent on DNA glycosylases namely: formamidopyrimidine (Fpg/MutM/Fapy), endonucleaseVIII (Nei) and endonucleaseIII (Nth) with Nei being structurally similar to Fpg but functionally similar to Nth. Bioinformatics analysis of the genome sequences of Mtb and its non-pathogenic relative Mycobacterium smegmatis (Msm) identified a unique duplication of Fpg and Nei glycosylases and a single nth gene in the same chromosomal context in both organisms. Previously, it has been shown that the lack of Fpg/Nei glycosylases in Msm display no differences in growth and survival under normal and oxidative stress conditions with no increase in spontaneous mutation rates as compared to the parental strain, suggesting that nth maybe significant for mycobacterial genome maintenance. Hence, in this study the nth gene was site specifically inactivated by homologous recombination in the parental Msm strain and in selected combinatorial mutant strains deficient in the Fpg/Nei glycosylases. Loss of the nth allele in the panel of mutants was genotypically confirmed by PCR and southern blot analyses. Inactivation of the nth gene did not affect the in vitro growth of the mutant strains under normal culture conditions. Interestingly, UV induced DNA damage of the single nth mutant resulted in a dramatic increase in mutation frequency that was not observed in any of the mutants. The progressive loss of fpg, nei and nth genes showed exaggerated reduced survival under oxidative stress. The subsequent deletion of nth in mutants deficient in fpg/nei resulted in a dramatic increase in spontaneous mutation rates and frequencies, implying that nth is integral for the repair of both spontaneous and induced DNA damage. Undoubtedly, these results indicate that Msm nth encoding the Nth glycosylase is involved in DNA repair and has anti-mutator properties. Furthermore, nth together with fpg and nei is part of a robust DNA repair system that maintains the integrity of the mycobacterial genome.Item Genotypic and phenotypic heterogeneity of Mycobacterium tuberculosis recovered from patients with pulmonary disease involving drug-resistant tuberculosis(2012) Axcell, AmandaGenetic heterogeneity of Mycobacterium tuberculosis demonstrating mixed infections or affecting single strains has been previously described. A single sputum culture from five patients with drug-resistant tuberculosis treated at Sizwe Hospital was analysed in-depth for genotypic and phenotypic heterogeneity. IS6110-based restriction fragment length polymorphism (RFLP) was performed on 20 colonies from each sputum for detection of mixed infections and clonal heterogeneity. No mixed infections were found, but IS6110-RFLP-linked clonal heterogeneity was observed in one patient. Drug susceptibility testing (DST) and sequencing of nine drug-resistance-associated genes performed on a total of 99 colonies from the five patients failed to show genotypic hetero-resistance. On DST, however, discordant rifampicin resistance findings were encountered in one patient. Minimal inhibitory concentrations performed on these colonies were close to the rifampicin critical concentration used for resistance determination, suggesting failure of the BACTEC MGIT 960 assay to reliably determine rifampicin susceptibility in strains with borderline resistance.