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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

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.
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ppat-1004446-g005: 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.

Mentions: In the ER lumen, β2M associates with MHC class I molecules to form class I complexes which are then loaded with peptide and transported to the cell surface for antigen presentation to CD8+ T cells. It is possible that once translocated to the ER, ESAT-6:CFP-10 can interact and sequester β2M and thereby reduce the availability of free β2M to form complex with HLA class I molecules. In such situations, the surface levels of both MHC and β2M molecules are likely to be decreased. Expectedly, when THP-1 macrophages were incubated with recombinant ESAT-6:CFP-10 protein at two different concentrations [17], [25], surface β2M levels were found to be decreased in a concentration dependent manner (Figure 5A and Figure S6). Interestingly, no significant changes in the surface expression of β2M were noticed in the presence of mutant ESAT-6ΔC:CFP-10 complex or CFP-10 alone (Figure 5A), indicating that suppression of β2M surface expression was possibly due to its sequestration by the intact C-terminal region of ESAT-6. To rule out the possibility that the reduction in surface β2M levels was due to toxicity of the ESAT-6:CFP-10 complex, an MTT viability assay was performed with THP-1 macrophages treated with ESAT-6:CFP-10. We found that the viability of the ESAT-6:CFP-10 treated cells was not affected (Figure S7). Another possibility is that incubation with ESAT-6:CFP-10 may have caused general trafficking defects to reduce the surface expression of β2M. To rule out this, we examined the surface expression of other cell surface markers like Mac-1, TLR4, MHC-II and CD14 using flow cytometry. Levels of all these molecules were found to remain unchanged in ESAT-6:CFP-10 treated cells (Figure S8). ESAT-6:CFP-10 also did not affect β2M expression at the protein and mRNA level (Figure S9). These data indicate that the reduction of surface β2M levels was possibly due to physical sequestration of β2M by ESAT-6 inside the ER. M. tuberculosis may escape from the phagosome to the host cytoplasm of infected cells in an RD1 dependent manner and secrete ESAT-6:CFP-10 directly into the cytoplasm of infected cells [26] which may find its way into the ER lumen to interact with the resident β2M. To test this hypothesis, we over-expressed FLAG- or GFP-tagged ESAT-6 in cells in an attempt to mimic physiological conditions where ESAT-6 is secreted directly into the cytosol. We transfected HEK-293 cells with pcDNA 3.1(+)-FLAG-esat-6 and studied localization of ESAT-6 (stained with anti-FLAG Ab) in ER by confocal microscopy and was found to be present in the calnexin-positive ER compartments (Figure 5B) suggesting that intracellular ESAT-6 can also find its way into the ER. We also performed co-immunoprecipitation studies in cells over-expressing full length ESAT-6 or ESAT-6ΔC cloned with an N-terminal EGFP tag in pEGFP-C1. ESAT-6 and β2M complexes could be pulled down from the whole cell lysates of HEK-293 cells over-expressing pEGFP-C1-esat-6 but not from cells transfected with pEGFP-C1 vector alone or pEGFP-C1-esat-6ΔC (Figure 5C). Also, we were able to pull down the ESAT-6:β2M complex from the enriched ER fraction of HEK-293 cells transfected with pEGFP-C1-esat-6 but not from cells transfected with the vector alone, indicating that ESAT-6:β2M complex was present inside the ER (Figure 5D). These data suggest that intracellular ESAT-6 is not only able to enter the ER but also can interact with the ER-resident β2M. We next examined, whether such interaction of ESAT-6 with β2M in ER has any effect on the surface β2M levels. Therefore, THP-1 cells were nucleofected and HEK-293 cells were transfected with pEGFP-C1, pEGFP-C1-esat-6 or pEGFP-C1-esat-6ΔC. Nucleofection efficiency for THP-1 was found to be ∼30% and transfection efficiency for HEK-293 was found to be ∼50%. Surface β2M expression on GFP positive cells was measured by flow cytometry using PE conjugated anti-human β2M Ab. It was observed that the intracellular expression of ESAT-6 resulted in reduction of surface β2M levels in THP-1 (Figure 5E) and HEK-293 (Figure 5F) cells when compared to the cells transfected with pEGFP-C1 or pEGFP-esat-6ΔC.


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)

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.
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ppat-1004446-g005: 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.
Mentions: In the ER lumen, β2M associates with MHC class I molecules to form class I complexes which are then loaded with peptide and transported to the cell surface for antigen presentation to CD8+ T cells. It is possible that once translocated to the ER, ESAT-6:CFP-10 can interact and sequester β2M and thereby reduce the availability of free β2M to form complex with HLA class I molecules. In such situations, the surface levels of both MHC and β2M molecules are likely to be decreased. Expectedly, when THP-1 macrophages were incubated with recombinant ESAT-6:CFP-10 protein at two different concentrations [17], [25], surface β2M levels were found to be decreased in a concentration dependent manner (Figure 5A and Figure S6). Interestingly, no significant changes in the surface expression of β2M were noticed in the presence of mutant ESAT-6ΔC:CFP-10 complex or CFP-10 alone (Figure 5A), indicating that suppression of β2M surface expression was possibly due to its sequestration by the intact C-terminal region of ESAT-6. To rule out the possibility that the reduction in surface β2M levels was due to toxicity of the ESAT-6:CFP-10 complex, an MTT viability assay was performed with THP-1 macrophages treated with ESAT-6:CFP-10. We found that the viability of the ESAT-6:CFP-10 treated cells was not affected (Figure S7). Another possibility is that incubation with ESAT-6:CFP-10 may have caused general trafficking defects to reduce the surface expression of β2M. To rule out this, we examined the surface expression of other cell surface markers like Mac-1, TLR4, MHC-II and CD14 using flow cytometry. Levels of all these molecules were found to remain unchanged in ESAT-6:CFP-10 treated cells (Figure S8). ESAT-6:CFP-10 also did not affect β2M expression at the protein and mRNA level (Figure S9). These data indicate that the reduction of surface β2M levels was possibly due to physical sequestration of β2M by ESAT-6 inside the ER. M. tuberculosis may escape from the phagosome to the host cytoplasm of infected cells in an RD1 dependent manner and secrete ESAT-6:CFP-10 directly into the cytoplasm of infected cells [26] which may find its way into the ER lumen to interact with the resident β2M. To test this hypothesis, we over-expressed FLAG- or GFP-tagged ESAT-6 in cells in an attempt to mimic physiological conditions where ESAT-6 is secreted directly into the cytosol. We transfected HEK-293 cells with pcDNA 3.1(+)-FLAG-esat-6 and studied localization of ESAT-6 (stained with anti-FLAG Ab) in ER by confocal microscopy and was found to be present in the calnexin-positive ER compartments (Figure 5B) suggesting that intracellular ESAT-6 can also find its way into the ER. We also performed co-immunoprecipitation studies in cells over-expressing full length ESAT-6 or ESAT-6ΔC cloned with an N-terminal EGFP tag in pEGFP-C1. ESAT-6 and β2M complexes could be pulled down from the whole cell lysates of HEK-293 cells over-expressing pEGFP-C1-esat-6 but not from cells transfected with pEGFP-C1 vector alone or pEGFP-C1-esat-6ΔC (Figure 5C). Also, we were able to pull down the ESAT-6:β2M complex from the enriched ER fraction of HEK-293 cells transfected with pEGFP-C1-esat-6 but not from cells transfected with the vector alone, indicating that ESAT-6:β2M complex was present inside the ER (Figure 5D). These data suggest that intracellular ESAT-6 is not only able to enter the ER but also can interact with the ER-resident β2M. We next examined, whether such interaction of ESAT-6 with β2M in ER has any effect on the surface β2M levels. Therefore, THP-1 cells were nucleofected and HEK-293 cells were transfected with pEGFP-C1, pEGFP-C1-esat-6 or pEGFP-C1-esat-6ΔC. Nucleofection efficiency for THP-1 was found to be ∼30% and transfection efficiency for HEK-293 was found to be ∼50%. Surface β2M expression on GFP positive cells was measured by flow cytometry using PE conjugated anti-human β2M Ab. It was observed that the intracellular expression of ESAT-6 resulted in reduction of surface β2M levels in THP-1 (Figure 5E) and HEK-293 (Figure 5F) cells when compared to the cells transfected with pEGFP-C1 or pEGFP-esat-6ΔC.

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