A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy February 2013 / Mycobacterium tuberculosis is presented with environmental host assaults that damage its DNA during infection. Tubercle bacilli possess mechanisms to protect against moststresses imposed by the host, including genotoxic stress. However, tolerance of DNA lesions that have escaped the normal repair processes requires the function of specialist DNA polymerases that can introduce mutations during translesion synthesis (replication by-pass), thus leading to damage-induced mutagenesis. Mycobacteria employ a novel DNA polymerase, DnaE2, for DNA damage tolerance and induced mutagenesis. DnaE2 belongs to the C-family of DNA polymerases, which are known to replicate DNA with high fidelity, and has been implicated in virulence and the emergence of rifampicin resistance of M. tuberculosis in vivo. In this study, DnaE2 was shown to function in the same pathway as two accessory proteins, ImuB and ImuA’, for damage tolerance and induced mutagenesis in mycobacteria. In this system, DnaE2 performs the polymerase function in translesion synthesis whereas ImuB is a cryptic Y-family DNA polymerase that lacks critical active site residues. It contains a β-clamp binding motif that allows interaction with the β-clamp and presumably enables DnaE2 and ImuA’ to access the replication fork. ImuB has a C-terminal region extending from the β-clamp binding motif which contains disordered regions that allow the interaction with other proteins and is important for function. ImuA’ is also essential for damage tolerance and induced mutagenesis but its function remains unknown. This protein is structurally similar to Escherichia coli RecA protein in the N-terminus and the middle domain, but it has a distinct C-terminus that was shown to be important for the interaction with ImuB. The essential replicative, C-family polymerase, DnaE1, was shown to be upregulated in response to DNA damage and was also shown to interact with ImuB. To explore the possibility that other proteins are involved in this pathway, ImuB was Cterminally tagged for use as bait in pull-down experiments in M. smegmatis. However, introduction of the tag disrupted ImuB function, further reinforcing the importance of the Cterminal region of ImuB for the function of this protein, presumably via protein-protein interactions. In contrast, a variant of ImuA’ which was N-terminally tagged was shown to retain functionality; however, experiments using this protein as a bait for pull-down proved to be unsuccessful. Proteomic analysis of wild type M. smegmatis, a dnaE2 deletion mutant and complemented derivative was carried out on cells exposed to the same conditions as used in the pull-down assay. Base excision repair (BER) components were identified in this analysis, but did not detect ImuB and ImuA’, suggesting that the levels of expression of these proteins were comparatively lower under the conditions tested resulting in failure of the pull-down experiment. Finally, numerous attempts were made to express and purify recombinant forms of ImuB and ImuA’ in E. coli for use in structural studies. Both proteins were expressed in the soluble and insoluble fractions; however the levels of soluble protein were low, and as a result, purified protein preparations could not be obtained.

Mycobacterial infections are responsible for some of the most well known disease, including Tuberculosis. Reported cases of infections caused by mycobacteria that are becoming increasingly resistant to traditional antibiotics are on the increase. This calls for a new approach in developing new drugs that can act on novel antimicrobial targets. One such alternative involves the use of bacteriophages and the study of how they interact with their hosts. Their diversity also suggests that there are many different phage-host interactions acting on multiple targets that are currently still unknown. Eight phages were isolated and characterized. Genomic libraries were constructed on four of these phages and screened for antimicrobial activities using Rhodococcus erythropolis. Six clones were further analyzed, and 15 ORFs were predicted with 8 ORFs being assigned functions. These genes with similarity to proteins in the database suggest that they are involved in membrane integrity and DNA metabolism. These clones were further tested on Saccharomyces cerevisiae to determine whether they have any effects on eukaryotes. The lack of inhibition in S. cerevisiae suggests these phage products are confined to act only in bacteria after millions of years of co-evolution with their host counterparts, and further studies into these genes will continue to shed light on bacterial genomics.

BCG is the only licensed human vaccine currently available against TB. Derived from a virulent strain of M. bovis, the vaccine was thought to have struck a balance between reduced virulence and preserved immunogenicity. Nowadays, BCG vaccine strains used in different countries and vaccination programs show clear variations in their genomes and immune protective properties. The aim of this study was to characterize the proteomic profile on Mycobacterium bovis and five BCG strains Pasteur, Tokyo, Danish, Phipps and Birkhaug by Tandem Mass Tag® (TMT®)-labeling quantitative proteomic approach. In total, 420 proteins were identified and 377 of them were quantitated for their relative abundance. We reported the number and relationship of differential expressed proteins in BCG strains compared to M. bovis and investigated their functions by bioinformatics analysis. Several interesting up-regulated and down-regulated protein targets were found. The identified proteins and their quantitative expression profiles provide a basis for further understanding of the cellular biology of M. bovis and BCG vaccine strains, and hopefully would assist in the design of better anti-TB vaccine and drugs.

No description available.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1951. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [31]-33).

Thesis (M.Med.Sc.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 42-57). Also available in print.

The ability of Escherichia coli to use the expression signals of mycobacterial genes was tested by inserting fragments of M. bovis BCG DNA into the E. coli promoter-probe plasmid pKK232-8. Comparison with the promoter activity achieved following insertion of restriction fragments of the E. coli host into. pKK232-8 revealed that a significant proportion of M. bovis BCG promoters were functional in E. coli. These results confirmed the suitability of E. coli as a host for the cloning and expression of mycobacterial genes. Using a variety of E. coli cloning vectors (pBR322, pUC13, EMBL4 and gtll), M. bovis BCG and M. leprae DM gene libraries were prepared. Recombinant M. bovis BCG clones were screened with rabbit antiserum and clones expressing M. bovis BCG antigens were identified. A pBR322/M. bovis BCG clone, expressing a 65KD molecule, was isolated and this antigen was shown to be cross-reactive with a 65KD M. leprae antigen. Recombinant gtll clones, expressing antigenic M. bovis BCG molecules, were also detected and a partial DM sequence was determined for one of these molecules. Moreover, recombinant gtll clones expressing (i) an 85KD biotinylated M. leprae molecule and (ii) an 85KD biotinylated M. bovis BCG molecule were also detected. In an attempt to test the feasibility of diagnosing leprosy by the presence of antibodies to specific antigens, antisera samples from leprosy patients and their contacts were screened for antibodies to mycobacterial antigens. Although only a small number of antisera were tested, a number of candidate antigens were identified.

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.

Yes / Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

No description available.