Mycobacterium tuberculosis is a major human pathogen causing significant human suffering worldwide. As the leading cause of death from a single bacterial infection, it is imperative to understand the biology, physiology and pathogenicity of the organism. Improved diagnostics, therapeutics and prophylactics are urgently required. M. tuberculosis exhibits certain features which contribute to its pathogenicity and potential to establish latent infections. Among these, the lipid-rich cell wall plays a critical part in interaction with the host, being the first line of defence against immune attack. The primary aim of this exploratory project is to define the physiological role of one of the critical, major cell wall components, lipoarabinomannan (LAM) in the tubercle bacillus. It has not been possible to study LAM in the context of the whole cell previously, since LAM negative mutants have not been isolated and we have recently shown that this is due to its essentiality for bacterial survival. Although, mutants cannot be constructed, we have engineered a strain of M. tuberculosis which expresses EmbC, one of the key enzymes required for LAM biosynthesis, at a reduced level compared to wild-type cells, the consequence of which is that LAM of a smaller size is produced. We will determine what effect changes in LAM production have on resistance to a wide range of compounds including ethambutol, DNA damaging agents and reactive oxygen and nitrogen species. A lack of EmbC is lethal in M. tuberculosis, but reduced expression is tolerated. We will determine how low EmbC expression can be reduced before bacterial survival is compromised by constructing a strain expressing embC from a controllable, titratable promoter. Subsequently, we will conduct phenotypic assays to determine the physiological consequences of EmbC depletion. Particular attention will be paid to the size and structure of LAM synthesized and resistance to damaging agents and stresses. The data generated will provide a picture of the physiological role of LAM in the bacterial cell and provide a platform for further work investigating pathogenicity.
Mycobacterium tuberculosis is the causative agent of human tuberculosis, a devastating infectious disease, which kills nearly 2 million and infects more than 8 million people each year. There is an urgent unmet need for new therapeutic agents and an increased understanding of the disease process. This proposal aims to increase our understanding of one of the key bacterial effectors of pathogenesis and define its role in bacterial physiology.
| Korkegian, Aaron; Roberts, David M; Blair, Rachel et al. (2014) Mutations in the essential arabinosyltransferase EmbC lead to alterations in Mycobacterium tuberculosis lipoarabinomannan. J Biol Chem 289:35172-81 |
