The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

Abstract

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.

Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis via Bacterial Antigens

“…This might be a result of variable expression of ESAT-6 and CFP-10 in different strains of Mtb (31, 32). A recent study has also demonstrated that the host protein χ 2 -microglobulin can bind ESAT-6 and mask a tryptic cleavage site (33) that is required to generate the ESAT-6 1900.95 m/z peptide recognized by our NanoDisk-MS assay (34). The differences in CFP-10 and ESAT-6 detection rates may be due to a combination of these 2 factors.…”

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring

“…Serum CFP-10 was detectable at lower concentrations than ESAT-6, perhaps owing to variable expression levels of ESAT-6 and CFP-10 in different strains of Mtb (40,41). However, host β2 microglobulin also has been reported to bind ESAT-6 to mask a tryptic cleavage site (amino acids 90-95) required for the 1,900.95 m/z ESAT-6 peptide (16,42), and thus may inhibit ESAT-6 cleavage to decrease the apparent serum ESAT-6 levels in our assay.…”

“…likely owing to the epitope-masking effects of host proteins (16) and homology with related antigens of several nontuberculous mycobacteria (NTM) (17,18).…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…Thereby, ESAT6 was shown to interact with beta-2-microglobulin in the ER, leading to inhibited loading of antigen-derived peptides on the MHC-I complex [221]. This is in line with other reports demonstrating a role for ESAT6 in antigen presentation on MHC-II [222].…”