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Characterisation of the structural properties and features of M. tuberculosis complex proteins linked to tuberculosis pathogenesis

By Sami Abdullah D. Al-Harbi


The genome of Mycobacterium tuberculosis encodes for 11 pairs of Esx family proteins such as EsxA/EsxB and EsxO/EsxP that are located in pairs within the genome. Despite the clear importance of the Esx family proteins in mycobacterial virulence and pathogenesis, the precise molecular functions and mechanisms of action for these proteins remain unknown. Initially expression vectors carrying EsxO and EsxP were constructed and used to express these proteins as inclusion products. The inclusion bodies of both proteins were successfully resolubilized and co-refolded. The final purification step by gel filtration chromatography shows that these proteins form a tight 1:1 heterodimeric complex. Analysis using circular dichroism (CD) spectroscopy of the purified refolded complex showed that it contained a high helical content (53%). The complex showed a significant resistance to heat-induced denaturation with co-operative denaturation observed that indicates a stable folded structure. In addition, significant chemical shift dispersion was seen in 1D1H NMR of the EsxO/EsxP complex, which clearly indicates a folded structure. \ud Previous studies have shown that the EsxA/EsxB complex binds specifically to the surface of monocyte and macrophage cells. Fluorescence microscopy studies described here show specific binding of the EsxO/EsxP complex to the surface of monocyte and macrophage cells, suggesting that EsxA/EsxB and EsxO/EsxP complexes bind to specific target but distinct targets on the surface of host cells, which suggests possible roles in pathogen-host cell signalling. Further work, I investigated whether exposure to the EsxO/EsxP complex results in changes in host cell motility or gene expression. Analysis of macrophage motility over period of eight hours revealed that neither EsxA/EsxB nor EsxO/EsxP has any effect on the motility of macrophages. In addition, microarray analysis was used to identify any changes in the gene expression profile of monocyte cells when exposed to the EsxO/EsxP complex. Interestingly, after 30 minutes exposure to EsxO/EsxP complex only 6 genes showed significant change in expression, but three of these are involved in regulation of chromatin structure. After 2 hours exposure to EsxO/EsxP complex still only small numbers of genes showed significant changes, but no clustering to specific biological processes was apparent. The observation of specific cell surface binding of the EsxO/EsxP complex strongly suggests role in signaling, however the precise function of the complex remains to be elucidated

Publisher: University of Leicester
Year: 2011
OAI identifier:

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