The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage
Figure 5
Exogenous addition of ESAT-6:CFP-10 complex or transient expression of ESAT-6 downregulates expression of surface β2M.
(A) PMA-differentiated THP-1 macrophages were treated with ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 or CFP-10 protein for 2 hours and stained with PE conjugated anti-human β2M Ab for measuring surface expression of β2M by flow cytometry. Median fluorescence intensities of the β2M levels of various groups were calculated and the results are shown as mean ± SD of 3 different experiments. (B) HEK-293 cells were transfected with either pcDNA 3.1 (+)-FLAG control plasmid or pcDNA 3.1 (+)-FLAG-esat-6 and after 20–24 hours, cells were fixed, permeabilized and stained with anti-calnexin Ab followed by Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red) to visualize the endoplasmic reticulum and anti-FLAG Ab followed by Alexa Fluor 488 conjugated secondary anti-mouse Ab (green) to visualize intracellular ESAT-6. Nucleus was visualized by DAPI staining (blue). Cells were observed under confocal microscope. (C) Lysates prepared from the HEK-293 cells transfected with either pEGFP-C1 or pEGFP-C1-esat-6 or pEGFP-CI-esat-6ΔC were incubated with anti-GFP Ab bound to protein A/G agarose beads. Immunoprecipitated complexes (Lanes 4–6) were separated on a 15% SDS-PAGE and transferred to a nitrocellulose membrane which was probed with anti-β2M Ab. About 10% of the lysates were loaded as input control (Lanes 1–3). (D) HEK-293 cells were transfected with either pEGFP-C1 or pEGFP-C1-esat-6 and after 20–24 hours, cells were used to prepare enriched rough endoplasmic reticulum fractions (RER). Equal amounts of proteins extracted from the enriched RER fractions were incubated with anti-GFP Ab bound to protein A/G agarose beads (Lanes 3 and 4). Immunoprecipitated complexes were separated on a 15% SDS-PAGE and transferred to a nitrocellulose membrane and was probed with anti-β2M Ab. About 10% of the lysates of enriched RER fractions (Lanes 1 and 2) were loaded as input controls. (E) THP-1 cells were nucleofected and (F) HEK-293 cells were transfected with either pEGFP-C1 or pEGFP-C1-esat-6 or pEGFP-CI-esat-6ΔC and after 20–24 hours, cells were stained with PE conjugated anti-human β2M Ab and β2M expression on the cell surface was measured by flow cytometry for EGFP-positive cells. Results are representative of three different experiments.