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.

At the author’s request, the abstract has been removed due to the confidential nature of the thesis. It will be added once the embargo period has passed.

Tuberculosis (TB) is the leading cause of death from a single infectious agent worldwide and HIV-1 co-infection is the leading cause of susceptibility to tuberculosis. Sub-Saharan Africa has a high incidence of TB-HIV-1 coinfection and the risk of TB in HIV-1 infected people is increased at all stages of the infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. By studying the protective, ART induced immune reconstitution in HIV infected individuals sensitised by Mycobacterium tuberculosis (Mtb), we can identify correlates of protection against tuberculosis in the form of transcriptomic, soluble or cellular biomarkers. This thesis focuses on characterising Mtb-specific reconstituting CD4 T cells as well as soluble and transcriptomic markers in HIV infected persons, sensitised by Mtb, by analysing samples collected longitudinally during 6 months of ART. Analysis of peripheral blood mononuclear cells by 14-colour flow cytometry revealed the proportion and numbers of central memory CD4+ T cells significantly expanded in HIV infected persons on ART, while the proportion and numbers of effector memory and terminally differentiated effector CD4+ T cells decreased significantly. Additionally we noted a significant decrease in the proportion of activated CD4+ T cells, and IL-2 single producing CD4+ T cells in HIV infected persons at 6 months of ART, while polyfunctional Mtb specific CD4+ T cells secreting IFN-γ, IL-2 and TNF-α simultaneously, proportionally increased. Analysis of soluble markers in the plasma of HIV infected persons revealed an overall decrease in pro-inflammatory cytokines during 6 months of ART. A significant decrease in IFN-γ, IL-1α, IL-1β, IL-6, IL-17A and TNF-α was observed, and concentrations of these cytokines fell towards those observed in HIV uninfected persons. Transcriptomic analyses of 30 genes normalized to 3 different housekeeping genes, showed an overall increase in the expression of T cell memory specific genes, illustrating the regeneration of the memory T cell pool in HIV infected adults on ART. Larger number of central memory specific genes showed increased expression when normalised to at least two housekeeping genes, as compared to effector memory specific genes. These results support the reconstitution of central memory CD4 T-cell specific response at 6 months of ART. Our data provides insight into the reconstituting immune response to latent TB infection in the context of HIV infection and identifies potential correlates of decreased susceptibility to TB. We also show decreasing soluble and cellular factors indicative of decreasing immune activation in HIV infected persons receiving ART.

A dissertation submitted to the Faculty of Health science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg 2013 / The 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.

No description available.

The purpose of this study was to characterize Johne’s disease in Mississippi cattle. Nine hundred eighteen animals from 23 sale barns in Mississippi were tested for Mycobacterium avium subspecies paratuberculosis (MAP). Ten milliliters of blood and 4-10 grams of feces were collected from cattle over two years of age presented to the attending auction veterinarian. Information obtained at the time of collection included the animal’s sex, type, and reproductive status. Serum samples were screened by an enzyme-linked immunosorbent assay (ELISA) for MAP. Shedding status was determined using polymerase chain reaction (PCR) on corresponding fecal samples. Overall, 17.3% (4/23) of sale barns had at least one animal sero-positive for MAP and 0.54% (5/918) were PCR positive. These results show a Johne’s disease prevalence similar to the estimate of 0.4% of animals infected found by the USDA NAHMS Beef ‘97 study, emphasizing the need for continued prevention and control practices.