Genomic variation and gene expression of clinical isolates of Mycobacterium tuberculosis
Members of the Mycobacterium tuberculosis complex, M. tuberculosis, M. bovis, M. microti, M. canettii and M. africanum, share over 99.9% identity at the DNA level. However, several recent studies have shown that there is considerably more genomic variation within the species when the organisation of the entire genome is considered, rather than primary sequences of individual genes. Therefore, to uncover the genomic variation within the species, one of the aims of this project was to compare the genome of several strains of M. tuberculosis, M. africanum, M. bovis (human and cattle isolates) and M. bovis BCG (Pasteur) using the M. tuberculosis H37Rv DNA microarray. The Beijing family of strains within the M. tuberculosis complex has been frequently associated with outbreaks of tuberculosis. Furthermore, several recent studies have shown that there are considerable selective advantages of the Beijing strains such as multidrug-resistance, higher adaptability to exposure to antituberculosis drugs and more infectivity in patients in countries such as China and Russia. The aim of this project was to understand the molecular basis for differences in virulence and infectivity amongst M. tuberculosis Beijing strains. The specific aims of the project were: (i) To investigate gene expression in response to stress conditions in the Beijing 94-1576 and Beijing 94-1707 strains. (ii) To investigate in vivo phenotypes of the Beijing 94-1576 and Beijing 94- 1707 strains. Comparative genomics was used to identify deletions of the RD1-RD14 regions as defined in M. bovis BCG. Regions of difference (RD) were used to reveal an evolutionary pathway for members of the M. tuberculosis complex and from this study, the presence of RD9 in one of the M. bovis isolated from cattle revealed a divergence from the published evolutionary pathway. It was anticipated that the selection of the M. tuberculosis strains from different sources might help uncover associations between genotype and host specificity in addition to exploring the scope of genotypic diversity. The M. tuberculosis isolates from India and different geographical areas such as Tanzania, Vietnam and Argentina did show a wide variation in the number, size and distribution of deletions. The Beijing strains showed a specific pattern of genomic variability with each having four large deletions compared to H37Rv. The genotypic characteristics of the Beijing family are (i) absence of the TbDl region, (ii) polymorphisms in the Rv3135 gene, the a/G463CTG (Leu) and gyrA95ACC (Thr) alleles and (iii) transposase insertion in the dnaA-dnaN and NTF-1 regions. In this study, the Beijing genotyping was performed and did confirm that the 94-1576 and 94-1707 strains were Beijing strains. The DNA microarray technique is only informative in detecting deletions that are present in the test strains compared with the reference strain H37Rv. To overcome this limitation, the subtractive hybridisation technique was applied and was able to identify a diverse polyketide synthase gene in the Beijing 94-1576 and Beijing 94-1707 strains. A study on gene expression in response to environmental stress conditions was performed to understand the influence of genome differences on gene regulation in H37Rv, Beijing 94-1576 and Beijing 94-1707. Using this, it proved possible to identify a number of significant differentially expressed genes involved (i) in oxidative and low pH stress for H37Rv, (ii) in oxidative and low pH stress for Beijing 94-1576, (iii) low pH and nitrosative stress for Beijing 94-1707. Finally, a number of experiments were done to investigate phenotypic differences between H37Rv, Beijing 94-1576 and Beijing 94-1707. This was done with a view to understand why the Beijing strains have become so successful in spreading across the world. In the initial stage of growth in the Balb/C mice, H37Rv, Beijing 94-1576 and Beijing 94-1707 grew progressively until day 28 before differences were seen between the strains. Infection in the human monocytic cell line THP-1 showed that the mycobacterial load of both Beijing 94-1576 and Beijing 94-1707 was higher than H37Rv. However, in C57BL/6 mice and murine bone marrow derived macrophages, the mycobacterial load of Beijing 94-1707 was significantly lower than H37Rv and Beijing 94-1576.