The burgeoning tuberculosis epidemic worldwide is mainly due to the reactivation of old latent tuberculosis infection. South Africa is rated as one of the countries with the worst tuberculosis epidemic and the population that is mostly affected is the mineworkers. Reports suggest that reactivation of latent tuberculosis infection is responsible for these high tuberculosis rates in this population. Various risk factors for reactivation of latent TB have been identified and include silicosis, diabetes mellitus, immunosuppressive drug therapy, Human Immunodeficiency Virus infection and malnutrition. The aim of this study was to determine whether there is any relationship between exposure to low levels of ionizing radiation and reactivation tuberculosis by evaluating the effects of radiation on dormant mycobacteria in vitro. The Wayne in vitro dormancy model was used to induce dormancy in Mycobacterium smegmatis and Mycobacterium bovis BCG. Dormant pGFM-11- transformed and non-transformed cultures were then exposed to 0.1, 1 and 5Gray Cobalt-60 radiation. The radiation effects were evaluated using viable counts, the bacillary adenosine triphosphate assay and quantification of the green fluorescent protein expression using flow cytometry after 24 and 72 hours of radiation exposure. Exponential phase cultures treated in exactly the same way as the dormant cultures together with the cultures that were not exposed to any radiation served as controls. Dormancy was successfully induced as determined by the sensitivity of the dormant cultures to metronidazole, resistance to isoniazid and assumption of synchronous replication on dilution into oxygen-rich medium. Subsequent to exposure to Cobalt-60 radiation, the dormant cultures were sensitive to metronidazole and resistant to isoniazid and the inverse was observed in irradiated exponential phase cultures. The results suggested that both dormant and exponential phase cultures of the tested mycobacteria retained their antibiotic susceptibility pattern and thus were not affected by Cobalt-60 radiation. It was concluded that the doses of Cobalt-60 radiation used in this study did not cause the reactivation of in vitro dormant mycobacterial strains tested. / Dr. H. Abrahamse Mrs. J.V. Hind

Confidential, not available. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

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M.Sc. / The global tuberculosis (TB) epidemic has resulted in the development of numerous methods for the enumeration and viability assessment of Mycobacterium tuberculosis (M.tb), as these methods play a key role in TB management and research. In this study the methods of quantitative culture (CFU), the microplate alamar blue assay (MABA), flow cytometry, the green fluorescent protein microplate assay (GFPMA) and quantitative PCR were investigated and compared for the enumeration and viability assessment of mycobacteria in culture. The MABA and the GFPMA were applied to the enumeration and viability assessment of mycobacteria post-infection. Quantitative culture was found to be simple and low in cost but was lengthy. The MABA, an economic and quick assay, was more sensitive for high mycobacterial concentrations. The flow cytometric enumeration of fluorescent mycobacteria was rapid and sensitive, but was dependent on access to a flow cytometer and therefore was costly. Flow cytometry facilitated enumeration but was limited concerning viability assessment. The GFPMA was a simple, rapid and cost effective assay. However, decreased sensitivity was observed for low mycobacterial concentrations. Quantitative PCR, although high in cost, was sensitive and rapid. The MABA and the GFPMA were useful for the enumeration of mycobacteria post-infection, with the former being the more sensitive method. This study serves as a reference of the methods available for the enumeration and viability assessment of M.tb. The advantages and disadvantages established for each of the methods investigated in this study enables an informed selection of the most appropriate method for a specific objective and research environment.

Although it is more than 100 years since Robert Koch discovered the tubercle bacillus, and more than 40 years since effective chemotherapy became available, the incidence of tuberculosis is increasing in much of the developing world and has recently re-emerged as a public health problem in industrialized countries. This problem is compounded by the increase in host susceptibility to tuberculosis caused by co-infection with HIV (Human Immunodeficiency Virus) and the emergence of Mycobacterium tuberculosis strains that are resistant to the front-line drugs. These factors highlight the urgent need for development of new drug classes to counter the threat posed by tuberculosis. The purpose of the present study was to develop a mouse model for Mycobacterium tuberculosis with the aim of determining the antimycobacterial activity of medicinal plants used by traditional doctors to treat tuberculosis in South Africa. Furthermore, the toxic effects of these medicinal plants in uninfected mice were determined. A field trip to the Northern Cape, Western Cape, Eastern Cape and Free State provinces was undertaken and medicinal plants used by traditional doctors to treat tuberculosis or its symptoms were collected, identified and examined for their therapeutic effects against Mycobacterium tuberculosis, determined using the mouse model. In addition, the effects of medicinal plants on the production of cytokines and granuloma formation in infected mice were examined. Six-to-ten week old C57BL/6 mice were infected with 107 viable Mycobacterium tuberculosis H37Rv strain by an aerosol exposure model. Bacterial growth was monitored by sacrificing infected but untreated mice at day 1, week 2 and week 4. Treatment with medicinal plant extracts was started 2 weeks after infection and continued for 2 weeks. An INH-RIF combination was used as positive controls. The bacterial load in infected but untreated mice increased by 1 log unit each week for 2 to 3 weeks. Bacterial loads were not detected in INH-RIF treated mice after 2 weeks of treatment. Treatment of mice with high doses of plant extracts was toxic. None of the tested medicinal plant extracts showed any activity against Mycobacterium tuberculosis. The production of IL-12 at week 4 was suppressed/ decreased when plant extract A was given at different concentrations. The bacterial loads in the lungs of the plant extract A treated mice was higher than that of the untreated mice (p < 0.005). Histological analysis of the lungs also revealed a high number of bacilli and increased size of the formed granuloma. In conclusion, the selected plant extracts obtained by water extraction exhibited no anti-tuberculosis activity in the laboratory mouse model for Mycobacterium tuberculosis infection. Furthermore, it was also shown that some plant extracts suppressed the production of IL-12, which plays an important role in the host's defense against Mycobacterium tuberculosis. However, further work is required to test if treatment for longer periods exhibits antituberculous activity.

Tuberculosis (TB) is the leading killer globally owing to an infectious disease. There is consequently an urgent need to develop novel TB drugs and shorter regimens to treat the causative agent, Mycobacterium tuberculosis, an imperative which demands the identification of new drug targets in essential mycobacterial pathways. To that end, the work presented in this dissertation aimed to functionally characterize ribF, an essential gene in the mycobacterial riboflavin (RF; vitamin B2) biosynthetic pathway. Given the role of RF as a core component of the essential flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it was hypothesized that silencing ribF would disrupt the biosynthesis of all flavoproteins, crippling numerous (essential) processes within the organism. Moreover, based on previous observations in Bacillus subtilis, it was predicted that the mycobacterial ribF homolog might play a role in regulating the rib operon (comprising a cluster of RF pathway genes) – either directly by binding to the FMN riboswitch, or indirectly through the production of FMN from RF, in turn enabling riboswitch-mediated repression of downstream genes. CRISPR interference (CRISPRi) technology was used to generate an anhydrotetracycline (ATc)-inducible ribF hypomorph of M. smegmatis, a widely exploited mycobacterial model. Consistent with other organisms, ribF was shown to be essential for in vitro growth of M. smegmatis: CRISPRi-mediated depletion of ribF was bacteriostatic, resulting in a 10-fold growth inhibition in liquid media and corresponding to no reduction (0 log-fold change) in colony forming units (CFU). Moreover, targeted metabolomic analyses revealed that ribF depletion was associated with accumulation of 6,7-Dimethylribityllumazine (DMRL), suggesting that the disruption of RibF function blocked conversion of RF to the essential cofactors, FMN and FAD, in turn inhibiting cell growth. Notably, the lethality of ribF depletion could not be complemented chemically by exogenous supplementation of growth media with RF, FMN or FAD. Downregulation of ribF also caused enhanced susceptibility to the known cell wall-targeting agent, vancomycin, but not to the putative RibF domain inhibitor, thonzonium bromide, suggesting an alternative mechanism of action or impaired bacillary permeation. In summary, these data support the inferred essentiality of ribF in mycobacteria, in turn supporting future work which aims to target this enzyme for new TB drug discovery.

Background: Despite the development of new drugs and success of social programs, tuberculosis remains a leading cause of mortality. This burden falls disproportionately on developing countries where the high burden of HIV has a potentiating e↵ect, but may soon return to areas where it was previously brought under control as resistant strains continue to emerge. In the Western Cape, two closely related strains of the Beijing family have been isolated that provide an opportunity to study virulence in a system with relatively little noise. The aim of this project was to identify the cause of the altered virulence displayed between the two strains, and describe how the di↵erences between the two genomes contributed to the phenotypic di↵erences. Results: GenGraph allows for the creation of graph genomes, and facilitated the creation of a pan-transcriptome that allowed for the mapping of gene annotations between isolates. This allowed for the mapping of reads to a more suitable Beijing family reference while interpreting the results with annotations from the H37Rv reference. We generated expression and target profiles for the known sRNA, and identified a large number of novel sRNA. Transcriptomic data from 4 di↵erent growth conditions was integrated with this sRNA data as well as variant data using the Cell pipeline. From this data we identified multiple sets of genes linked to copper sensing in MTB, including the di↵erentially expressed MoCo operon. Increasing evidence that macrophages use copper to poison bacteria trapped in their phagosomes provides the link to virulence and pathogenicity. Conclusions: Through the integration of data from multiple data types we were able to elucidate the most probable cause of the altered virulence found between the two isolates in this study. We developed reusable tools and pipelines, and noted a large number of undescribed sRNA expressed in these isolates. The identification of the copper response as a chief contributor to the phenotype increases both our understanding of the isolates, and the role of the element in infection. These results will be key in guiding further investigation of the variant linked genes to identify those linked to copper homeostasis or response.

Cholesterol has been shown to play important role in the pathogenesis and persistence of intracellular pathogens. Here, we modulate host cholesterol biosynthesis pathway using pharmacological agent statins, which are reversible inhibitors of HMG†CoA reductase enzyme. The aim of the study was to investigate the role of statins in inducing host protective responses against intracellular pathogens. We report reduced growth of Listeria monocytogenes (LM) and Mycobacterium tuberculosis (Mtb) in murine macrophages. We show prominent immunomodulatory activity induced by statins, mainly increased phagosomal maturation and autophagy resulting in decreased bacterial growth in macrophages. Subsequently, statin†treated mice showed decrease in bacterial loads, accompanied by reduced histopathology in the acute phase of infection during listeriosis and tuberculosis. Furthermore, we found decreased growth of Mtb in peripheral blood mononuclear cells (PBMC) and monocyte†derived macrophages (MDM) isolated from patients with familial hypercholesterolemia (FH) on statin therapy when compared to healthy subjects. Together, our results show that statins induces protection against Mtb in murine macrophages, mice and human mononuclear cells and monocyte†derived macrophages.

South Africa is one of the countries with the highest burden of Mycobacterium tuberculosis (TB) in the world. The fact that adult patients diagnosed with TB frequently do not attend their primary healthcare clinics after discharge from hospital for continued treatment remains a challenge for public health in South Africa. This qualitative study employed semi-structured interviews, focus group discussions and observations explored the experiences of patients, their families, healthcare workers and policy makers, with continuity of TB care following diagnosis in hospital. The key research question was what factors were shaping patients’ attendance at primary healthcare clinics following TB diagnosis and start of treatment in tertiary and district hospitals. Sub questions were: how did patients diagnosed with TB interpret and act upon their diagnosis and treatment at the tertiary/district hospital? What roles did patients play in the discharge process? What were their home circumstances and experiences at the clinics they were referred to, regarding their registration and follow-up plan? What were the perceptions of patients, healthcare workers and policy makers on what influences patients’ attendance/non-attendance at clinics? The objective of this study was to contribute to our understanding of patients’ experiences and perceptions of treatment of TB and how services to patients could be improved to enhance better continuity of care. I drew on a three-fold theoretical framework: patient-centred care, Foucault’s concept of the 'medical gaze’ and social determinants of health. My study built upon previous and ongoing research on the topic of continuity of care for TB in Cape Town. I argued that problems in the provision of TB services to hospital patients could be understood as failures of the services at the hospital to achieve some of the core components of patient-centered care. Furthermore, I argued that better systems for following-up patients from the hospitals to their homes and clinics would provide more understanding of the challenges patients faced when they have been referred from a tertiary or district hospital to continue with their treatment. Insights gained from qualitatively following patients from diagnosis to discharge and their home circumstances helped to better understand the problem South Africa faced with continuity of care for TB treatment.

Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host.