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Mycobacterium tuberculosis, the causative agent of tuberculosis, can manipulate host cell death pathways as virulent strains inhibit apoptosis to protect its replication niche and induce necrosis as a mechanism of escape. In vitro studies revealed that similar to lytic viruses, M. tuberculosis has the ability to induce cytolysis in macrophages when it reaches an intracellular burden of ~25 bacilli. Base on this finding, we proposed the burst size hypothesis that states when M. tuberculosis invades a macrophage at a low multiplicity of infection it replicates to a burst size triggering necrosis to escape the cell and infect naïve nearby phagocytes, propagating the spread of infection. The first part of this study investigated if the in vitro observations of M. tuberculosis cytolysis were relevant to cell death of infected phagocytes during pulmonary tuberculosis in vivo. Mice infected with a low dose of M. tuberculosis revealed during TB disease, the major host cell shifted from one type of phagocyte to another. Enumeration of intracellular bacilli from infected lung cells revealed the predictions of the hypothesis were confirmed by the distribution of bacillary loads across the population of infected phagocytes. Heavily burdened cells appeared nonviable sharing distinctive features similar to infected macrophages from in vitro studies. Collectively, the data indicates that M. tuberculosis triggers necrosis in mononuclear cells when its number reaches the threshold burst size. The previous study showed during the period of logarithmic bacterial expansion, neutrophils were the primary host cell for M. tuberculosis coinciding with the timeframe of the highest rate of burst size necrosis. The second part of this study examined this link by infecting mice with one of four different M. tuberculosis strains ranging in virulence. Mice infected with the most virulent strain had the highest bacterial burden and elicited the greatest number of infected neutrophils with the most extensive lung inflammation and greater accounts of cell death. Treating these mice with a bacteriostatic agent decreased the bacterial load and infected neutrophils in a dose-dependent manner indicating necrosis induced by virulent M. tuberculosis recruited neutrophils to the lungs. Infected neutrophils can serve as a biomarker in tuberculosis as evidenced by poorly controlled infection and increased severity of lung immune pathology.

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Thesis (MPH.) -- University of Limpopo, 2022 / Background: Tuberculosis(TB) and Human Immunodeficiency virus(HIV) continues to be a public health concern globally. There is no data on TB outcomes on HIV programme outcome in Lebowakgomo hospital of Limpopo Province. The main objective of this study was to determine the TB treatment outcomes in TB/HIV co-infected people at Lebowakgomo hospital in Limpopo Province. Methodology: A quantitative retrospective design was used in the study in which a sample size of 180 patients’s files who are 18 years and above and TB/HIV co-infected were reviewed. A self-designed data collection tool was used to collect data. The tool covered variables such as age, gender, HIV status, CD4 cell count, type TB, duration on TB treatment and the outcome. Data was analysed using the STATA statistical software version 12 for Windows (STATA Corporation, College Station, Texas). Results: The majority of records were age group 35-44 years at 32%. There was a statistical significance differences (p˂0.001) between males and females in relation to age groups. TB treatment success rate was 68.9% and mortality 16.9%. Females were more likely to complete TB treatment successfully than males. Overall age, gender, previous TB infections, TB type, duration on ART and CD4 Count were not significantly associated with treatment outcomes amongst TB/HIV co-infected people. Conclusion: This study has revealed that TB treatment success rate in HIV co-infected is lower (68.3%) than the WHO target of 85%.

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No description available.

La tuberculose (TB) est une maladie mortelle qui frappe en grande partie les pays en développement. Près d’un quart de la population serait touché par la TB dans sa forme active ou latente. L’émergence de la résistance bactérienne rend ce problème de santé publique encore plus important. Certaines souches multirésistantes (MDR-TB) et extensivement résistantes (XDR- TB) sont en circulation dans certaines zones géographiques plus à risque. En collaboration avec l’Institut Pasteur de Madagascar, les objectifs de ce travail étaient premièrement de brosser le portrait global des mutations résistance aux antituberculeux circulant à Madagascar, et deuxièmement, d’effectuer une analyse comparative entre une méthode de séquençage ciblée (Deeplex—MycTB) et une méthode de séquençage du génome complet pour l’antibiogramme génotypique de la TB. L’analyse a été réalisée avec des souches MDR-TB ainsi que des souches sensibles aux antituberculeux en provenance de Madagascar. Les outils bio-informatiques Mykrobe et Clockwork ainsi que le test de séquençage ciblé Deeplex Myc- TB® ont été utilisés. Nos résultats ont permis de dresser la liste exhaustive des mutations de résistance circulantes à Madagascar entre 2012 et 2021 en plus d’évaluer la performance relative de deux méthodes de séquençage pour l’antibiogramme génotypique. / Tuberculosis (TB) is a deadly disease predominantly affecting developing countries. Close to one quarter of the population would be affected by TB in its active or latent form. The emergence of bacterial resistance has made this public health issue even more important. Multi-drug resistant (MDR-TB) and extensively drug resistant (XDR-TB) strains are circulating in some high-risk areas. With the collaboration with the Institute Pasteur of Madagascar, the goals of this project was first to describe the drug resistance mutations circulating in Madagascar, and second, to perform a comparative analysis between a targeted sequencing method (Deeplex—MycTB) and a whole genome sequencing method for TB genotypic drug susceptibility testing methods. Our analyses generated the comprehensive list of circulating resistance mutations in Madagascar between 2012 and 2021 and allowed to evaluate the relative performance of two sequencing methods for genotypic drug susceptibility testing.

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

Protein synthesis is a process by which organisms manufacture their proteins that perform various cellular activities either alone or in combination with other similar or different molecules. In eubacteria, protein synthesis proceeds at a rate of around 15 amino acids per second. The ribosomes, charged tRNAs and mRNAs can be considered as the core components of protein synthesis system which, in addition, involves a panel of non-ribosomal proteins that regulate the speed, specificity and accuracy of the process. Peptidyl-tRNA hydrolase (Pth) and ribosome recycling factor (RRF) are two such non-ribosomal proteins involved in protein synthesis. These two proteins are essential for eubacterial survival and the work reported in this thesis involves structural characterization of these two proteins from the bacterial pathogen, Mycobacterium tuberculosis. The protein structures were solved using established techniques of protein crystallography. Hanging drop vapour diffusion method and crystallization under oil using microbatch plates were the methods employed for protein crystallization. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator in all the cases. The data were processed using DENZO and MOSFLM. The structures were solved by molecular replacement method using the program PHASER. Structure refinements were carried out using programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, CHIMERA, and PYMOL were used for structure validation and analysis of the refined structures. Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA that has dropped off from ribosome before reaching the stop codon, in order to avoid the toxicity resulting from peptidyl-tRNA accumulation and to free the tRNA to make it available for further rounds in protein synthesis. To begin with, the structure of the enzyme from M. tuberculosis (MtPth) was determined in three crystal forms. This structure and the structure of the same enzyme from Escherichia coli (EcPth) in its crystal differ substantially on account of the binding of the C-terminus of the E.coli enzyme to the peptide binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in open conformation when the enzyme is in the free state as in the crystals of MtPth. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the EcPth crystal mimics the peptide-bound conformation of the enzyme. The peptide stretch involving a loop and a helix, referred to earlier, now closes on the bound peptide. Concurrently, a gate connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of the two residues. Thus, the crystal structure of MtPth when compared with that of EcPth, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. A discrepancy between the X-ray results and NMR results, which subsequently became available, led to X-ray studies on new crystal forms of the enzyme. The results of these studies and those of the enzyme from different sources that became available, confirmed the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding site and tRNA binding site. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movement is not, however, observed in the NMR structure of MtPth. The discrepancy between the X-ray and NMR structures of MtPth in relation to the functionally important plasticity of the molecule, referred to earlier, also led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures are available appears to confirm this correlation. In consonance with the reported results of the investigation in cellular components and aqueous solutions, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important for function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule. After termination of protein synthesis at the stop codon, the ribosome remains as a post-termination complex (PoTC), consisting of the 30S and the 50S subunits, mRNA and a deacylated tRNA. This complex has to be disassembled so that the ribosome is available for the next round of translation initiation. Ribosome recycling factor (RRF) binds to ribosome and in concert with elongation factor G (EF.G), performs the recycling of ribosome that results in disassembly of PoTC. The structure of this L-shaped protein with two domains connected by a hinge, from Mycobacterium tuberculosis (MtRRF) was solved previously in our laboratory. The relative movement of domains lies at the heart of RRF function. Three salt bridges were hypothesized to reduce the flexibility of MtRRF when compared to the protein from E.coli (EcRRF), which has only one such salt bridge. Out of these three bridges, two are between domain 1 and domain 2, whereas the third is between the hinge region and the C-terminus of the molecule. These salt bridges were disrupted with appropriate mutations and the structure and activity of the mutants and their ability to complement EcRRF were explored. An inactive C-terminal deletion mutant of MtRRF was also studied. Major, but different, structural changes were observed in the C-terminal deletion mutant and the mutant involving the hinge region. Unlike the wild type protein and the other mutants, the hinge mutant complements EcRRF. This appears to result from the increased mobility of the domains in the mutant, as evidenced by the results of librational analysis. In addition to the work on PTH and RRF, the author was involved during the period of studentship in carrying out X-ray studies of crystalline complexes involving amino acids and carboxylic acids, which is described in the Appendix of the thesis. The complexes studied are that of tartaric acid with arginine and lysine.