Doctoral Degrees (Molecular Biology and Human Genetics)
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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by Author "Bruiners, Natalie"
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- ItemInvestigating the Human-M. tuberculosis interactome to identify the host targets of ESAT-6 and other mycobacterial antigens(Stellenbosch : Stellenbosch University, 2012-12) Bruiners, Natalie; Gey van Pittius, Nicolaas Claudius; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics.ENGLISH ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis, is an intracellular pathogen that secretes virulence factors, namely ESAT-6 and CFP-10, as substrates of the ESX-1 secretion system. It is hypothesised that these substrates interact with host proteins in a targeted manner in order to elicit a required immune response, and they have been shown to be involved in processes related to pro-inflammatory responses, necrosis, apoptosis, membrane lysis and cytolysis. However, the biological function of ESX-1 substrates during host-pathogen interactions remains poorly and incompletely understood. Therefore, the present study was designed to gain insight into the role of the ESX-1 secretion system substrates in host-pathogen interactions and to identify how M. tuberculosis mediates the response of the human host. In this study, a cDNA yeast two-hybrid library was constructed from human lung mRNA, to identify mycobacterial-host protein-protein interactions that occur within the lung alveoli. The ESX-1 secretion system substrates, ESAT-6 and CFP-10, were cloned in-frame into the pGBKT7 vector, which was used in the yeast two-hybrid system to screen the lung cDNA library in Saccharomyces cerevisiae. The ESAT-6 and CFP-10 screens identified 79 and 19 positive colonies, respectively. Of the total number of clones characterised, only two in-frame inserts were identified with the ESAT-6 screen, corresponding to the human proteins filamin A and complement component 1, q subcomponent, A chain (C1QA). In addition, the screen with CFP-10 also identified C1QA as binding partner. Subsequent in vitro and in vivo experiments were unable to confirm the putative interactions of C1QA with ESAT-6 and CFP-10. However, the interaction between filamin A and ESAT-6 was demonstrated and confirmed by both in vivo co-localisation and co-immunoprecipitation. Furthermore, the degradation of filamin A in the presence of ESAT-6 was shown to be reflective of cytoskeleton remodelling and the induction of cell death. The work presented here suggests that as ESAT-6 gains access to the cytosol, it initiates cell death by inducing destabilisation of the cytoskeleton cell structure. This may possibly be driven by the interaction of ESAT-6 and filamin A. Finally, we also initiated an investigation of the identified putative binding partners (filamin A and C1QA) as possible genetic markers for genetic susceptibility studies to tuberculosis. A case-control analysis was performed involving 604 cases, of which 109 were Tuberculous Meningitis (TBM), and 486 were controls from the South African Coloured (SAC) population within the Ravensmead-Uitsig catchment area. The results of this analysis demonstrated a novel association of a regulatory variant (rs587585) located upstream of the C1QA gene and demonstrated an increasing trend towards increased values in tuberculosis patients with the associated genotype. This study has contributed significantly to our understanding of human-mycobacterial hostpathogen protein-protein interactions and has opened the way for future studies further exploring the consequences and function of the identified ESAT-6-filamin A interaction. It has also led to the identification of a novel genetic association with tuberculosis. Finally, it demonstrates the usefulness of the yeast two-hybrid system to identify potential proteinprotein (host-pathogen) interactions that can lead to additional important and exciting research.