The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage
Figure 2
ESAT-6 with a deletion of six C-terminal amino acids (ESAT-6ΔC) fails to interact with β2M.
(A) Yeast strain AH109 transformed with ESAT-6/ESAT-6ΔC bait along with β2M prey plasmid were streaked on interaction selection QDO plates (SD/–Ade/–His/–Leu/–Trp) along with the controls and monitored the growth resulting from positive interactions. (B) ESAT-6 or ESAT-6ΔC expressed as GST fusion protein was incubated with THP-1 lysate and precipitated using glutathione-agarose. Presence of β2M in the precipitated complexes was detected by Western blotting using anti-β2M Ab. (C) Recombinant His-tagged ESAT-6 or His-tagged ESAT-6ΔC protein was mixed and incubated with THP-1 cell extracts and β2M was immunoprecipitated using mouse anti-human β2M Ab and protein A/G beads. Presence of ESAT-6/ESAT-6ΔC and β2M in the immunoprecipitated complexes were detected by Western blotting using rabbit anti-His Ab and rabbit anti-human β2M Ab respectively. Lanes 1 and 2 are input controls. (D) THP-1 macrophage whole cell extract was mixed and incubated with recombinant ESAT-6:CFP-10 or mutant ESAT-6ΔC:CFP-10 or CFP-10 protein. β2M was immunoprecipitated using protein A/G bound mouse anti-human β2M Ab and the immune complexes were subjected to Western blotting using rabbit anti-His Ab to detect ESAT-6:CFP-10 or mutant ESAT-6ΔC:CFP-10 or CFP-10. The lanes 1–3 are input controls for these recombinant proteins (upper panel). In the lower panel, the lanes 1–3 indicate the levels of β2M in the input controls as determined by Western blotting using rabbit anti-human β2M Ab.