Investigating the Human-M. tuberculosis interactome to identify the host targets of ESAT-6 and other mycobacterial antigens

[摘要] ENGLISH ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis, is an intracellularpathogen that secretes virulence factors, namely ESAT-6 and CFP-10, as substrates of theESX-1 secretion system. It is hypothesised that these substrates interact with host proteins in atargeted manner in order to elicit a required immune response, and they have been shown tobe involved in processes related to pro-inflammatory responses, necrosis, apoptosis,membrane lysis and cytolysis. However, the biological function of ESX-1 substrates duringhost-pathogen interactions remains poorly and incompletely understood. Therefore, thepresent study was designed to gain insight into the role of the ESX-1 secretion systemsubstrates in host-pathogen interactions and to identify how M. tuberculosis mediates theresponse of the human host.In this study, a cDNA yeast two-hybrid library was constructed from human lung mRNA, toidentify mycobacterial-host protein-protein interactions that occur within the lung alveoli. TheESX-1 secretion system substrates, ESAT-6 and CFP-10, were cloned in-frame into thepGBKT7 vector, which was used in the yeast two-hybrid system to screen the lung cDNAlibrary in Saccharomyces cerevisiae. The ESAT-6 and CFP-10 screens identified 79 and 19positive colonies, respectively. Of the total number of clones characterised, only two in-frameinserts were identified with the ESAT-6 screen, corresponding to the human proteins filaminA and complement component 1, q subcomponent, A chain (C1QA). In addition, the screenwith CFP-10 also identified C1QA as binding partner.Subsequent in vitro and in vivo experiments were unable to confirm the putative interactionsof C1QA with ESAT-6 and CFP-10. However, the interaction between filamin A and ESAT-6was 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 bereflective of cytoskeleton remodelling and the induction of cell death. The work presentedhere suggests that as ESAT-6 gains access to the cytosol, it initiates cell death by inducingdestabilisation of the cytoskeleton cell structure. This may possibly be driven by theinteraction 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 TuberculousMeningitis (TBM), and 486 were controls from the South African Coloured (SAC) populationwithin the Ravensmead-Uitsig catchment area. The results of this analysis demonstrated anovel association of a regulatory variant (rs587585) located upstream of the C1QA gene anddemonstrated an increasing trend towards increased values in tuberculosis patients with theassociated genotype.This study has contributed significantly to our understanding of human-mycobacterial hostpathogenprotein-protein interactions and has opened the way for future studies furtherexploring the consequences and function of the identified ESAT-6-filamin A interaction. Ithas also led to the identification of a novel genetic association with tuberculosis. Finally, itdemonstrates the usefulness of the yeast two-hybrid system to identify potential proteinprotein(host-pathogen) interactions that can lead to additional important and exciting research.

[发布机构] Stellenbosch University