Limits...
The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage.

Sreejit G, Ahmed A, Parveen N, Jha V, Valluri VL, Ghosh S, Mukhopadhyay S - PLoS Pathog. (2014)

Bottom Line: The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities.The C-terminal six amino acid residues (90-95) of ESAT-6 were found to be essential for this interaction.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad, India.

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214792&req=5

ppat-1004446-g006: 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.

Mentions: Beta-2-microglobulin remains associated with HLA class I, CD1 and HFE molecules on the cell surface. Any change in the levels of β2M would probably lead to changes in the cell surface expression of HLA class I, CD1 and HFE. As ESAT-6:CFP-10 treatment leads to reduction in surface expression of β2M (Figure 5), we next examined whether it has any effect on HLA class I expression. For this purpose, we utilized two different types of anti-HLA antibodies: HC-10 and W6/32. HC-10 is a monoclonal Ab that can detect only the free HLA-I heavy chain molecules not complexed with β2M [29]. Newly synthesized HLA molecules form a tri-molecular complex with β2M and antigenic peptide which is transported to the surface to present the peptide to its cognate T cell receptor (TCR) [30]. In contrast, it has been reported that heavy chains of MHC class I alleles can also progress to the cell surface alone without being associated with β2M. Such free MHC class I heavy chains are present normally on cells, especially in activated lymphocytes [31], dendritic cells and macrophages and have been found to be upregulated in patients suffering from spondyloarthropathy [32]. Interestingly, we observed an increased binding of HC-10 Ab in THP-1 macrophages treated with ESAT-6:CFP-10 but not with ESAT-6ΔC:CFP-10 (Figures 6A and 6B). Increased HC-10 staining upon addition of ESAT-6:CFP-10 was observed also by confocal microscopy where intracellular levels of the free HLA molecules can also be determined (Figure 6C). These data clearly suggest that the levels of β2M-free HLA class I molecules were increased on the surface as well as intracellularly after treatment with ESAT-6:CFP-10. Free HLA class I molecules are not only transported to the cell surface but a portion may also undergo proteasomal degradation since addition of MG-132 (a proteasomal inhibitor) to THP-1 macrophages resulted in increased HC-10 staining which was further intensified in the presence of ESAT-6:CFP-10 (Figures 6D and 6E). Therefore, it appears that once ESAT-6:CFP-10 complex sequesters β2M inside the ER, the number free HLA class I heavy chain molecules are increased due to less availability of free β2M molecules to associate with them. This would possibly explain the increase in HC-10 staining observed in the presence of ESAT-6:CFP-10.


The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage.

Sreejit G, Ahmed A, Parveen N, Jha V, Valluri VL, Ghosh S, Mukhopadhyay S - PLoS Pathog. (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214792&req=5

ppat-1004446-g006: 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.
Mentions: Beta-2-microglobulin remains associated with HLA class I, CD1 and HFE molecules on the cell surface. Any change in the levels of β2M would probably lead to changes in the cell surface expression of HLA class I, CD1 and HFE. As ESAT-6:CFP-10 treatment leads to reduction in surface expression of β2M (Figure 5), we next examined whether it has any effect on HLA class I expression. For this purpose, we utilized two different types of anti-HLA antibodies: HC-10 and W6/32. HC-10 is a monoclonal Ab that can detect only the free HLA-I heavy chain molecules not complexed with β2M [29]. Newly synthesized HLA molecules form a tri-molecular complex with β2M and antigenic peptide which is transported to the surface to present the peptide to its cognate T cell receptor (TCR) [30]. In contrast, it has been reported that heavy chains of MHC class I alleles can also progress to the cell surface alone without being associated with β2M. Such free MHC class I heavy chains are present normally on cells, especially in activated lymphocytes [31], dendritic cells and macrophages and have been found to be upregulated in patients suffering from spondyloarthropathy [32]. Interestingly, we observed an increased binding of HC-10 Ab in THP-1 macrophages treated with ESAT-6:CFP-10 but not with ESAT-6ΔC:CFP-10 (Figures 6A and 6B). Increased HC-10 staining upon addition of ESAT-6:CFP-10 was observed also by confocal microscopy where intracellular levels of the free HLA molecules can also be determined (Figure 6C). These data clearly suggest that the levels of β2M-free HLA class I molecules were increased on the surface as well as intracellularly after treatment with ESAT-6:CFP-10. Free HLA class I molecules are not only transported to the cell surface but a portion may also undergo proteasomal degradation since addition of MG-132 (a proteasomal inhibitor) to THP-1 macrophages resulted in increased HC-10 staining which was further intensified in the presence of ESAT-6:CFP-10 (Figures 6D and 6E). Therefore, it appears that once ESAT-6:CFP-10 complex sequesters β2M inside the ER, the number free HLA class I heavy chain molecules are increased due to less availability of free β2M molecules to associate with them. This would possibly explain the increase in HC-10 staining observed in the presence of ESAT-6:CFP-10.

Bottom Line: The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities.The C-terminal six amino acid residues (90-95) of ESAT-6 were found to be essential for this interaction.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad, India.

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.

No MeSH data available.


Related in: MedlinePlus