ABSTRACT
ESAT-6 is a small secreted protein of Mycobacterium tuberculosis involved in the ESAT-6 secretion system (ESX-1)-mediated virulence and pathogenesis. The protein interacts with ß2M, causing downregulation of MHC class I Ag presentation, which could be one of the mechanisms by which it favors increased survival of the bacilli inside the host. In an earlier study, we have shown that the C-terminal region of ESAT-6 is crucial for its interaction with ß2M. However, the interface of ß2M involved in interaction with ESAT-6 and detailed physicochemical changes associated with ESAT-6:ß2M complexation are not fully defined. In this study, using computational and site-directed mutagenesis studies, we demonstrate the presence of strong noncovalent hydrophobic interactions between ESAT-6 and ß2M in addition to the vital hydrogen bonding between the aspartate residue (Asp53) of ß2M and methionine (Met93) of ESAT-6. Docking-based high-throughput virtual screening followed by 16-point screening on microscale thermophoresis resulted in the identification of two potent inhibitors (SM09 and SM15) that mask the critical Met93 residue of ESAT-6 that is required for ESAT-6:ß2M interaction and could rescue cell surface expression of ß2M and HLA in human macrophages as well as MHC class I Ag presentation suppressed by ESAT-6 in peritoneal macrophages isolated from C57BL/6 mice. Both SM09 and SM15 significantly inhibited intracellular survival of M. tuberculosis in human macrophages. Further, we characterized the physicochemical properties involved in the ESAT-6:ß2M complexation, which may help in understanding host-pathogen interactions.
