Abstract
The ESAT-6 protein of Mycobacterium tuberculosis (M. tb) is an important structural and functional protein, which has been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen; however, how ESAT-6 protein interact with host protein is still unclear. In order to study the function of the M. tuberculosis protein ESAT-6 in the infection process, we searched for host proteins that interact with this secreted mycobacterial protein. Using a yeast two-hybrid system we identii¬ed the ADAM9 (a disintegrin and metalloprotease) protein as a candidate to interact with ESAT-6. This interaction was further coni¬rmed by protein overlay and surface plasmon resonance binding assay using recombinant ESAT-6 and ADAM9, and by GST pull-down analysis of the mycobacterial expressed ESAT-6 and ADAM9. The interaction domains were localized by yeast two-hybrid studies using truncated derivatives of ESAT-6 protein. The C-terminus of ESAT-6 binds to the ADAM9, Thus, the host protein ADAM9 represents a possible cellular receptor for the mycobacterial protein ESAT-6. This is the first report demonstrating the interaction of ADAM9 with a structural protein of M. tb. Key words: ESAT-6 protein, ADAM9 protein, Mycobacterium tuberculosis, yeast two-hybrid, GST pull-down assay, surface plasmon resonance binding
Highlights
Pathogenic mycobacteria, in particular Mycobacterium tuberculosis, the causative agent of tuberculosis, have the remarkable capacity to circumvent destruction within one of the most hostile cell types of a vertebrate host: the macrophage
4 of 34 clones survived all genetic tests and were considered to be genuine positive clone, DNA sequence analysis of the fragment revealed that the four cDNA fragments inserted have a high identity with four genes in the GeneBank database. One of these clones was identified as ADAM9 protein, As shown in Figure 3, the protein encoded by the pGAD-ADAM9 clones interacted with the ESAT-6 protein
By employing a series of biochemical and biophysical methods, we have firstly reported that ESAT-6 protein has a specific binding affinity to human ADAM9, and the further yeast twohybrid assay demonstrated that the C-terminus of ESAT6 probably contribute to the ESAT-6/human ADAM9 interaction
Summary
Pathogenic mycobacteria, in particular Mycobacterium tuberculosis, the causative agent of tuberculosis, have the remarkable capacity to circumvent destruction within one of the most hostile cell types of a vertebrate host: the macrophage. The ability of pathogenic mycobacteria to survive inside macrophages has been known for more than 30 years; yet, only recently have advances in molecular genetics, biochemistry, immunology, as well as global analysis of gene expression, started to unravel that M. tb infection modifies the gene expression of the host macrophage, which results in a highly sophisticated interaction between the pathogen and its host (James et al, 2007; Durrant et al, 2010) better understanding of the molecular mechanism of interaction between the pathogen and its host is important for effective control and prevention of TB (Yew et al, 2008; Rao et al, 2007). A knockout of ESAT-6 in Mycobacterium bovis results in decreased virulence of the pathogen (Wards et al, 2000), indicating that the two molecules may play important roles in immunopathogenesis and virulence, in this study, we use ESAT-6 protein as the bait to screen for interacting proteins from a human lung cDNA library by the yeast two-hybrid assay system
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