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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 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).
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ppat-1004446-g007: 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).

Mentions: On the other hand, when the surface expression of β2M-complexed HLA class I molecules was measured with the help of W6/32, (a monoclonal Ab that recognizes a conformation specific epitope on the HLA class I molecules only when associated with β2M [33], [34]) we observed a significant decrease in staining in ESAT-6:CFP-10-treated but not in ESAT-6ΔC:CFP-10-treated cells (Figures 7A and 7B). Also, a pull down assay with W6/32 yielded lesser amount of β2M in ESAT-6:CFP-10 treated cells, indicating that less amount of β2M was complexed with HLA class I molecules in these cells compared to untreated as well as those treated with ESAT-6ΔC:CFP-10 (Figure 7C). Data from Figures 6 and 7 indicate that ESAT6:CFP-10 can sequester free β2M in ER resulting in reduced HLA class I-β2M complex formation and consequently increasing the levels of free HLA class I heavy chain molecules.


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 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214792&req=5

ppat-1004446-g007: 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).
Mentions: On the other hand, when the surface expression of β2M-complexed HLA class I molecules was measured with the help of W6/32, (a monoclonal Ab that recognizes a conformation specific epitope on the HLA class I molecules only when associated with β2M [33], [34]) we observed a significant decrease in staining in ESAT-6:CFP-10-treated but not in ESAT-6ΔC:CFP-10-treated cells (Figures 7A and 7B). Also, a pull down assay with W6/32 yielded lesser amount of β2M in ESAT-6:CFP-10 treated cells, indicating that less amount of β2M was complexed with HLA class I molecules in these cells compared to untreated as well as those treated with ESAT-6ΔC:CFP-10 (Figure 7C). Data from Figures 6 and 7 indicate that ESAT6:CFP-10 can sequester free β2M in ER resulting in reduced HLA class I-β2M complex formation and consequently increasing the levels of free HLA class I heavy chain molecules.

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