Little is known about the survival mechanics of Mycobacterium tuberculosis within the human host. A number of genes are likely to be activated during infection, especially within macrophages, and these potential virulence determinants will be of great importance in the ultimate development of new vaccines and antimycobacterials. In an effort to identify some of these virulence determinants, a M.tuberculosis H37Rv gene promoter-probe library in a BCG host was constructed using an E.coli - mycobacterial shuttle vector with lacZ as the reporter gene. 4800 individual clones were arrayed in a 96 well microtitre(TM) format, enabling the testing of individual clones for promoter activity under a variety of environmental stressing conditions. Two separate in vitro screenings of the arrayed clones identified 53 clones that exhibited up-regulation of lacZ when stressed. 50 of these clones were bi-directionally sequenced to determine the cloned M.tuberculosis H37Rv DNA sequence. Identification of the location of the cloned DNA within the M.tuberculosis H37Rv genome identified 32 clones likely to containing potential promoter sequences. By measuring the levels of lacZ expression post-infection of macrophages, compared to in vitro expression, clones were identified which contained potential promoter sequences up-regulated within the macrophage. Quantitative Reverse Transcriptase-PCR was employed, using cDNA obtained from M.tuberculosis from infected and un-infected macrophages, to measure the levels of expression of 5 M.tuberculosis H37Rv genes identified as potentially up- regulated during infection. The results suggest that two of the five genes, genes Rv1265 and Rv2711, are indeed up-regulated during infection. The protein coded for by the gene Rv1265 has an unknown function whilst the gene Rv2711 encodes the protein IdeR, which is an iron-dependent repressor. Sequence and lacZ expression analysis identified four independently selected and screened clones which contained near identical sequences for the same promoter - that of the gene Rv0440 (groEL2). Gene Rv0440 codes for a 60KDa heat shock protein previously identified as up-regulated during infection. The identification of four independent clones containing the promoter for this gene validates this technology as a means of identifying M.tuberculosis H37Rv genes up-regulated during infection.

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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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The purpose of this investigation was to extract, separate, partially characterize and compare the individual phospholipids of the atypical mycobacteria.

PhD - Science / Specialised mechanisms have evolved in pathogenic bacteria to enable adaptation to hostile and fluctuating host environments. Inducible mutagenesis, in particular, has been implicated in the emergence of antibiotic- and stress-resistant mutants. This thesis examined mycobacterial DNA metabolism with specific emphasis on the roles of multiple Y-family polymerases in the evolution of inter-strain variation and drug resistance in M. tuberculosis. The contribution of the nrdZ-encoded class II ribonucleotide reductase (RNR) to the maintenance of dNTP pools for replication and repair under hypoxic conditions was also explored. In addition, the co-factor requirement of NrdZ prompted an investigation into the biosynthesis and transport of adenosylcobalamin (AdoCbl) in M. tuberculosis. The data suggest that the mycobacterial Y-polymerases are tightly regulated and restricted to specialised damage-free repair or replication restart. Disruptions in individual M. smegmatis mc2155 DinB (pol IV) homologues resulted in novel antibiotic-resistance polymorphisms that were suggestive of non-redundant function. In contrast, abrogation of all error-prone polymerase activity failed to impair long-term competitive survival of mc2155 in vitro. Similarly, heterologous overexpression of M. tuberculosis pol IV homologues did not increase spontaneous mutation rates in wild-type mc2155, or complement damage hypersensitivity. However, treatment of M. smegmatis with gyrase inhibitors confirmed the differential induction of pol IV homologues in response to replication stalling and demonstrated elevated rates of spontaneous mutagenesis as a result of GyrB inhibition. The class II RNR does not appear to play a significant role in mycobacterial pathogenesis. Specifically, NrdZ was unable to substitute for the class I RNR under aerobic conditions in vitro, and a M. tuberculosis ÄnrdZ deletion mutant was not impaired in its ability to adapt to hypoxia in vitro. Similarly, infection of immunocompetent mice suggested that nrdZ is not required for the survival or virulence of M. tuberculosis in vivo. Disruptions in genes required for AdoCbl and methionine biosynthesis revealed that complex regulatory functions govern mycobacterial methionine and AdoCbl homeostasis. Loss of early (cobK) or late (cobU) stage AdoCbl biosynthetic enzymes had no effect on the growth of M. tuberculosis H37Rv in vitro. In contrast, deletion of the B12-independent methionine synthase (metE) resulted in impaired growth on solid media that could be rescued by vitamin B12 but not Lmethionine supplementation, simultaneously demonstrating the ability of M. tuberculosis to transport exogenous vitamin B12. Significantly, double ÄcobU/ÄmetE and ÄcobK/ÄmetE deletion mutants in which all predicted methionine synthase activity was eliminated, were not impaired for growth in liquid minimal media, suggesting that M. tuberculosis H37Rv possesses alternative mechanisms for methionine generation. Finally, the attenuated virulence of the ÄcobU and ÄmetE deletion mutants in vivo in immunocompetent mice indicated the relevance of AdoCbl biosynthesis to mycobacterial pathogenesis.