Figure 1.
ESAT-6 protein interacts with β2M.
(A) Interaction between ESAT-6 and β2M in a yeast two-hybrid system was studied by mating yeast strain AH1109 transformed with bait plasmid pGBKT7-ESAT-6 with yeast strain Y187 transformed with a human leukocyte cDNA prey library on a synthetic dropout plate (–Ade/–His/–Leu/–Trp). Prey cDNA was amplified by PCR using primers encompassing the cDNA insert in pACT2 and sequenced and identified to be β2M. The AH109 yeast strain transformed with pGBKT7-ESAT-6 and pACT2-β2M shows Ade and His interaction reporter activation on QDO plates. AH109 transformed with pGBKT7 and pACT2-β2M was used as a negative control while AH109 transformed with pGBKT7-p53 and pGADT7-T was used as a positive control for the yeast two-hybrid screening. (B) Untagged β2M was cloned along with His-tagged ESAT-6 in the pETDuet vector system and was transformed into E. coli BL21 cells. The transformed cultures were induced with IPTG and the over-expressed proteins were purified using TALON resin. Purified proteins were separated on a 16% Tris-Tricine SDS-PAGE and visualized by silver staining. Lane 1 is a molecular weight marker. (C) β2M with no N-terminal signal sequence (ESAT-6:β2M NSS) and the full length β2M with the N-terminal signal sequence (ESAT-6:β2M WSS) were cloned in pETDuet vector along with 6× His-tagged ESAT-6. Clones were over-expressed in E. coli BL21. Protein expression was induced by addition of IPTG and over-expressed proteins were purified using metal affinity TALON resin. Purified proteins were separated on 16% Tris-Tricine SDS-PAGE and transferred onto nitrocellulose membranes and probed with anti-β2M Ab to detect β2M and anti-His Ab to detect ESAT-6. The bands corresponding to β2M (upper panel) and ESAT-6 (lower panel) were visualized by chemiluminescence. (D) GST pre-cleared THP-1 macrophage extract was mixed and incubated with Glutathione-agarose bead bound-GST or GST-ESAT-6 and washed. The bound proteins were eluted and resolved on a 16% Tris-Tricine SDS-PAGE gel and immunoblotted for β2M protein using rabbit anti-human β2M Ab and HRP conjugated anti-rabbit Ab. Blots were visualized by chemiluminescence. Whole cell extracts from THP-1 cells was used as positive control for β2M expression. (E) In another experiment, purified recombinant 6× His-tagged ESAT-6 was incubated with THP-1 macrophage lysate and immunoprecipitated (IP) with anti-His Ab bound to protein A/G agarose beads. Control immunoprecipitation was carried out without the addition of His-tagged ESAT-6 protein (IP control). The eluted protein mixture is resolved on a 16% Tris-Tricine SDS-PAGE gel and immunoblotted (IB) for β2M protein using a rabbit anti-human β2M Ab and HRP conjugated anti-rabbit secondary Ab and the blots were visualized by chemiluminescence. Lane 1 is input control. (F) Direct interaction of β2M with ESAT-6 was monitored using a BIACORE 3000 Biosensor where β2M was immobilized on the sensor chip and recombinant ESAT-6 at different concentrations was injected in the running buffer. The changes in the refraction index at the surface due to interactions between immobilised β2M and fluid phase ESAT-6 were detected and recorded as RU (Resonance Units). Curves generated from the RU trace were evaluated using a curve-fitting algorithm. ESAT-6 was found to bind specifically to β2M and no binding was observed in a control cell which did not have any immobilized β2M. Results are representative of three different experiments.
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.
Figure 3.
The ESAT-6:β2M complex is stable at high salt concentrations and low pH.
(A) Cobalt agarose bead-bound ESAT-6:β2M complex was washed with increasing concentration of 0.5 M NaCl (Lane 2), 1 M Nacl (Lane 3) and 2 M NaCl (Lane 4). Proteins bound to these beads were eluted by boiling in 1× PAGE loading dye and resolved on a 16% Tris-Tricine SDS-PAGE gel and visualized by Coomassie blue staining. Lane 1 shows the molecular weight marker. (B) Purified ESAT-6:β2M protein complex was dialyzed in acetate buffers of varying pH like pH 4.0 (Lanes, 2, 6 and 10), pH 5.0 (Lanes, 3, 7 and 11), pH 6 (Lanes, 4, 8 and 12) and pH 8.0 (Lanes, 5, 9 and 13). After dialysis, the protein complexes were cross-linked by glutaraldehyde treatment for 15, 20 and 30 minutes at 37°C and resolved on a 16% Tris-Tricine SDS-PAGE gel and visualized by silver staining. Protein molecular weight marker was loaded in Lane 1. Results are representative of three different experiments.
Figure 4.
Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER.
PMA-differentiated THP-1 macrophages were incubated with FITC-labelled ESAT-6:CFP-10 (green) either at 4°C (A) or 37°C (B) for about 120 minutes. Cells were then washed, fixed, permeabilised and stained for an ER marker calnexin using rabbit anti-calnexin Ab and Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red). In another set of experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal macrophages (E) were treated with FITC-labelled ESAT-6 or ESAT-6:CFP-10 (green) for about 100 minutes at 37°C and then incubated with ER-Tracker dye (blue) for another 20 minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different experiments.
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.
Figure 6.
Soluble ESAT-6:CFP-10 increases the levels of free HLA class I heavy chain molecules.
(A) PMA-differentiated THP-1 macrophages were treated with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each). After 2 hours, cells were washed and incubated with anti-HLA class I heavy chain mAb HC-10 followed by FITC conjugated anti-mouse secondary Ab. Surface expression of free HLA class I heavy chain molecules were studied by flow cytometry. Cells stained with appropriate isotype Ab were used as control. (B) Median fluorescence intensities of different experimental groups described in Figure 6A were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages pre-treated with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each) were fixed, permeabilized and stained with HC-10 Ab followed by Alexa Fluor 594 conjugated anti-mouse secondary Ab (red). Matching isotype Ab was used as control. The nucleus was visualized by DAPI staining (blue). The stained cells were observed under a confocal microscope. (D) In another set of experiments, THP-1 macrophages were pre-treated for 30 minutes with 5 µM MG-132 followed by incubation with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each) for 2 hours. Free HLA class I molecules on the cell surface were stained with HC-10 Ab followed by staining with FITC conjugated anti-mouse secondary Ab and studied by flow cytometry. Isotype-matched Ab was used as control. (E) THP-1 macrophages treated with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each) in the absence or presence MG-132 werefixed, permeabilized and stained with HC-10 Ab followed by Alexa Fluor 594 conjugated secondary anti-mouse Ab (red). Nucleus was stained with DAPI (blue) and cells were visualized under a confocal microscope. Data shown is representative of three independent experiments.
Figure 7.
Soluble ESAT-6:CFP-10 reduces surface levels of β2M-associated HLA class I molecules.
(A) PMA-differentiated THP-1 macrophages were treated with 12.5 µM of either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein for 2 hours. Cells were stained with (W6/32) mAb followed by FITC conjugated anti-mouse secondary Ab. Expression of surface β2M conjugated HLA class I molecules was studied by flow cytometry. Isotype-matched Ab was used as control. (B) Median fluorescence intensities of different experimental groups of Figure 7A were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages were either left untreated (control) or treated with 12.5 µM of ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10. After 2 hours, cell were harvested and lysates were prepared. Equal amount of protein from each experimental group was incubated with W6/32 mAb bound to protein A/G agarose. Isotype matched Ab was used as control. Pulled-down complexes (Lanes 5–8) were resolved on a 15% glycine SDS-PAGE and transferred onto a nitrocellulose membrane which was probed with anti-β2M Ab. About 10% of the lysate was used as input controls (Lanes 1–4, upper panel). Equal loading in the input samples was also confirmed by probing the input controls with anti-β-actin Ab (Lanes 1–4, lower panel).
Figure 8.
Soluble ESAT-6:CFP-10 complex inhibits presentation of the SIINFEKL peptide.
(A) OVA (10 mg/ml) was hypertonically loaded on to C57BL/6 mouse peritoneal macrophages pre-treated with 7.5 µM of ESAT-6/CFP-10 or ESAT-6:CFP-10ΔC. Presentation of SIINFEKL peptide derived from OVA on MHC-I is quantitated by FACS using an Ab that recognizes SIINFEKL bound to H-2Kb MHC-I and median fluorescence intensities of SIINFEKL presentation of different groups were calculated and the results are shown as mean ± SD of 3 different experiments. The OVA pulsed cells were stained with PE conjugated isotype-matched control Ab. (B) Responses of the MHC class I-restricted T cell line B3Z to peritoneal macrophages presenting hypertonically loaded cytoplasmic OVA. Pre-fixed macrophages pulsed with OVA antigen and BSA were used as controls. (C) C57BL/6 mouse peritoneal macrophages pre-treated with 7.5 µM of soluble ESAT-6/CFP-10 or ESAT-6:CFP-10ΔC were incubated with 1 mg/ml of exogenously added soluble OVA and analyzed for cross-presentation of SIINFEKL peptide on H-2Kb MHC-I by FACS using an Ab that recognises SIINFEKL bound to H-2Kb MHC-I. Median fluorescence intensities of SIINFEKL presentation of different groups were calculated and the results are shown as mean ± SD of 3 different experiments.
Figure 9.
Detection of ESAT-6:β2M complex in pleural fluid.
Pleural fluid samples from 7 pleural TB positive and 7 TB negative patients were subjected to sandwich ELISA. The amount of β2M (A) and ESAT-6:β2M complex (B) was measured and compared on the basis of absorbance at 492 nm between TB positive and TB negative patients.