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The Actinomycetes, to which the Mycobacterium genus belongs contains many pathogens, such as Mycobacterium tuberculosis, which can cause tuberculosis, so it has become a focus area in modern molecular biology research. Mycobacteria contain many proteins and regulatory factors, including tRNA, ncRNA and sRNA, which can help bacteria better adapt to the environment. Among them, 6C RNA is a stem-loop non-coding RNA, widely found in mycobacteria. According to previous studies, it may be involved in the rapid growth of mycobacteria. We aimed to clone the 6C RNA promoter region into the pIGn plasmid carrying lacZ reporter gene and transform the construct into Mycobacterium marinum, a close relative of M. tuberculosis. Then we analyzed the β-galactosidase activity of the transformed strain under different stress conditions to study the change of 6C RNA expression. At the same time, we recorded the growth curve and analyze expression changes of 6C RNA in the exponential growth phase and stationary phase of the transformed strain. In addition, we tried to clone the 6C RNA overexpression vector and to study the changes of gene expression at different growth stages, which will help us to better understand the role of 6C RNA in M. marinum.

Thiamine-phosphate kinase (or ATP:thiamine-phosphate phosphotransferase, ThiL) and homoserine (ThrB) kinases are essential to metabolism in Mycobacterium tuberculosis (Mtb). ThiL and ThrB respectively phosphorylate thiamine monophosphate (TMP) to thiamine diphosphate (TDP), the active form of vitamin B1, and L-homoserine to Ophosphohomoserine, critical to aspartate biosynthesis. In this study, ThiL and ThrB from Mtb were characterised structurally and functionally by producing the proteins recombinantly in E. coli. Proteins were purified by affinity, anion exchange and size exclusion chromatographies and purity checked by SDS-PAGE. ThiL and ThrB enzyme activities were confirmed and reaction products verified by high pressure liquid chromatography (HPLC). The crystal structure of ThiL was solved by molecular replacement using X-ray diffraction data. Functionally active ThiL, 36 kDa, produced hexagonal crystals belonging to space group P6122 with one monomer per asymmetric unit. Structurally it is related to ThiL from other organisms with minor structural deviations. Enzymatically active ThrB, 33 kDa, was crystallised. However, crystals failed to diffract Xrays to a suitable resolution. ThiL and ThrB could act as possible anti-TB drug targets against Mtb. / Dissertation (MSc)--University of Pretoria, 2017. / Biochemistry / MSc / Unrestricted

>Magister Scientiae - MSc / The generation of biomedical research data on the African continent is grow- ing, with numerous studies realizing the importance of African genetic diver- sity in discoveries of human origins and disease susceptibility. The decrease in costs to purchase and utilize such tools has enabled research groups to produce datasets of signi cant scienti c value. However, this success story has resulted in a new challenge for African Researchers and institutions. An increase in data scale and complexity has led to an imbalance of infrastructure and skills to manage, store and analyse this data. The lack of physical infrastructure has left genomic research on the continent lagging behind its counterparts abroad, drastically limiting the sharing of data and posing challenges for researchers wishing to explore secondary analysis, study veri cation and amalgamation. The scope of this project entailed the design and implementation of a proto- type genome archive to support the e ective use of data resources amongst researchers. The prototype consists of a web interface and storage backend for users to upload and browse projects, datasets and metadata stored in the archive. The server, middleware, database and server-side framework are components of the genome archive and form the software stack. The server component provides the shared resources such as network connectivity, le storage, security and metadata database. The database type implemented in storing the metadata relating to the sample les is a NoSQL database. This database is interfaced with the iRods middleware component which controls data being sent between the server, database and the Flask framework. The Flask framework which is based on the Python programming language, is the development platform of the archive web application. The Cognitive Walkthrough methodology was used to evaluate suitabil- ity of the software for its users. Results showed that the core conceptual model adopted by the prototype software is consistent and that actions available to the user are visible. Issues were raised pertaining to user feedback when per- forming tasks and metadata term meaning. The development of a continent wide genome archive for Africa is feasible by utilizing open source software and metadata standards to improve data discovery and reuse.

Mycobacterium tuberculosis (Mtb) remains a global challenge that has been worsened by the emergence of drug resistant strains of Mtb. We used publicly available Next Generation Sequencing (NGS) and drug susceptibility (DST) data to develop “Resistance sniffer”, an online software program for the rapid prediction of lineage and Mtb drug resistance. Based on the distribution of polymorphisms in the genomes of Mtb, we calculated the power of association between the polymorphisms in different clades of Mtb and resistance to 13 anti-TB drugs. Our data suggests that the development of drug resistance in Mtb is a stepwise process that involves the accumulation of polymorphisms in the Mtb genome. We carefully curated the polymorphisms based on their association powers to create a diagnostic key that captures patterns of these polymorphisms that can be used to predict lineage and drug resistance in Mtb. This diagnosis key was incorporated into the Resistance Sniffer tool, an online software program that we developed for the rapid diagnosis of drug resistance in Mtb. The tool was tested using sequence data from the South Africa Medical Research Council (SA-MRC). Our data suggests that the majority of the strains in SA may have been brought by the arrival of European settlers while the more resistant strains may have been introduced in the region by Asian travellers later on. We next sought to determine non-random associations between polymorphic sites in genomes of Mtb. Using the attributable risk (Ra) statistical methods, we distinguished between functional associations and associations that may have been due to genetic drift events for different Mtb clades. We then integrated the (Ra) data with drug susceptibility and annotation data to generate networks in Cytoscape 3.71. These networks were then used to infer evolutionary trajectories that drive the emergence and fixation of the drug resistant phenotype in different clades of Mtb. We demonstrate that strains from the Lineage 1.2 are associated with less complex functional associations compared to the strains from other clades such as the Asian and Euro-American clades. Our data also shows that the predisposition of strains from the Asian clades to develop multi-drug resistance may be attributed to a complex network of functional interactions of mutations in genes that are involved in several aspects of Mtb physiology such as cell wall modelling, lipid metabolism, stress response and DNA repair. / Dissertation (MSc)--University of Pretoria, 2019. / Biochemistry / MSc / Unrestricted

Philosophiae Doctor - PhD / Marine organisms including algae and bacteria are known to produce chemically diverse secondary metabolites for survival purposes in the marine environment. Scientists have identified some of these natural products as therapeutic agents including some antibiotics. Given the increase in the resistance of pathogenic microorganisms especially methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis to commonly prescribed antibiotics, researchers have turned towards exploiting marine natural products for new antibacterial compounds. Due to the proven success of finding bioactive compounds in the marine environment this study therefore aims to discover lead compounds against MRSA and Mycobacterium tuberculosis from two marine sources, the marine algae and the bacteria associated with marine invertebrates referred to as bacterial isolates. / 2024

This first detailed description of the pathology of tuberculosis, caused by Mycobacterium tuberculosis in springbok is reported. The springbok were part of a semi free-ranging herd kept on the grounds of iThemba Laboratory for Accelerator Based Science (LABS) in the Kuils River district of the Western Cape Province, South Africa. Of the 33 animals sampled, two animals had tuberculosis lesions. Mycobacterium tuberculosis was isolated from these two animals, as well as an animal that did not show tuberculosis pathology. The index case was an adult ewe that was presented for necropsy in a severely weakened condition. The ewe showed advanced miliary tuberculosis with marked macroscopic lesions in the lungs, pleura and respiratory lymph nodes. Limited sampling was done but microscopic tuberculosis lesions were found in almost all the organs sampled, and acid-fast bacilli were generally numerous. Six healthy rams were culled nine months later and a pilot study indicated miliary tuberculosis lesions in one ram, which again were macroscopically most prominent in the lungs, pleura and respiratory lymph nodes. Macroscopic lesions were also noted in the sternal, iliac, prefemoral and retropharyngeal lymph nodes. Microscopy in this animal revealed lesions in the macroscopically affected organs as well as numerous other lymph nodes, and suspected lesions occurred in the testicle and colon. Acid-fast bacilli were scarce to moderate in affected organs. Because of the miliary nature of the lesions in both affected animals, the route of infection could not be established conclusively. The lesions in most affected organs of both animals resembled classical tuberculous granulomas, viz. central caseous necrosis, with various degrees of calcification, surrounded by various numbers macrophages, epithelioid cells, multinucleated giant cells and lymphoplasmacells, and mild to moderate fibrous encapsulation. Necrotic lesions in the spleen, liver and kidney of the ewe were more disseminate and coagulative. A main study conducted on healthy culled animals 19 months after the pilot study failed to find any animal with tuberculosis lesions in the group of 25 sampled. These animals were all negative for mycobacteria via mycobacterial culture. The Interferon-gamma (INFg) assay was performed on all the animals of the pilot and main study but failed to identify the culture-positive animals and showed one false-positive reaction. / Dissertation (MMedVet (Pathology))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms.

It is often difficult to discern true mycobacterial infection from colonization due to Mycobacterium gordonae (. M. gordonae) since this organism is ubiquitous and is commonly an innocuous saprophyte. This study reports a rare case of caseating hilar adenopathy and pulmonary disease caused by M. gordonae in a patient with chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis (RA) on maintenance steroids and methotrexate. Pathologic exam and cultures of lymph node excision biopsy and bronchoalveolar lavage (BAL) confirmed the diagnosis.Triple antimycobacterial therapy with azithromycin, ethambutol and rifabutin was administered. The patient had significant clinical and radiologic improvement and follow-up cultures confirmed microbiologic cure.Mycobacterium gordonae can be a rare cause of significant pulmonary infection, and positive sputum or BAL cultures for M. gordonae should not be automatically discarded and considered as nonpathogenic contaminants or colonizing organisms, especially in immunocompromised hosts with comorbidities. A detailed review of the case and relevant literature is provided.

Studies of the ancient history of infectious diseases have been facilitated greatly by development of a succession of novel analytical methods. In particular, laboratory analytical methods that are based on high-throughput ancient deoxyribonucleic acid sequencing have received considerable attention in this respect. Even so, significant environmental caveats remain. There are many means by which microbes move through soil, often fairly readily. Thus, the depositional component of the postmortem environment, especially with respect to unshielded animal or human remains, is a fertile arena for many microbes that can contaminate archaeological specimens well after deposition and decay of soft tissues. The huge number of pathogenic and nonpathologic genera and species clearly dictate renewed interest and research into the long-term biological activities of soil-covered remains. In a tuberculosis context, we focus on various depositional concerns and limitations, such as contamination prior to archaeological discovery (perhaps many years prior), various means of microorganism movement in soil, the influence of these factors on differential diagnosis, and real hazards for misinterpretation of investigational results.