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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

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.
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ppat-1004446-g002: 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.

Mentions: The extreme C-terminus of ESAT-6 is a floppy, structurally ill defined region, away from the helical core and is not known to be involved in the interaction with CFP-10 [14]. Mutant M. tuberculosis expressing a truncated ESAT-6 having residues deleted from the C-terminal end was found to be attenuated. However, these mutant strains had a functional ESX-1 system with normal secretion of the ESAT-6:CFP-10 complex [13], [15] indicating that the residues at the C-terminal end of ESAT-6 are not required for a structural role in the formation of a heterodimer with CFP-10 but crucial for the virulence functions of ESAT-6. Therefore, we speculated that the free C-terminus of ESAT-6 is probably involved in the interaction with β2M and such an interaction is possibly critical for M. tuberculosis virulence [21]. Once again using the yeast two hybrid assay, we observed that deletion of the last 6 amino acids (valine, threonine, glycine, methionine, phenylalanine and alanine) from the C-terminal end of ESAT-6 (Figure S2) was sufficient to prevent the interaction of ESAT-6 and β2M (Figure 2A). To further validate the yeast two hybrid data, we used a C-terminally truncated ESAT-6 (ESAT-6ΔC) to carry out GST pull down assay. The GST-tagged full length ESAT-6 protein (ESAT-6-GST) as well as the GST-tagged ESAT-6ΔC (ESAT-6ΔC-GST) protein were incubated with THP-1 extract and the pulled down fractions were probed for the presence of β2M using anti-β2M Ab. It was found that only the full length ESAT-6 protein was able to interact with β2M (Figure 2B). To further confirm our observations, we carried out a Co-IP assay where THP-1 lysates were incubated with full length His-tagged ESAT-6 or His-tagged ESAT-6ΔC and β2M-complexes were immunoprecipitated using anti-β2M Ab. The immunoprecipitated complexes were resolved on SDS-PAGE and probed with either anti-His Ab to detect ESAT-6/ESAT-6ΔC (Figure 2C, upper panel) or anti-β2M Ab to detect β2M (Figure 2C, lower panel). The data shown in Figure 2C clearly demonstrate that anti-β2M antibody can immunoprecipitate the full length ESAT-6 (Figure 2C, Lane 3), but not the mutant ESAT-6ΔC (Figure 2C, Lane 4), indicating that the C-terminal (90–95) residues of ESAT-6 are important for its interaction with β2M. Lane 5 of Figure 2C is negative control where Protein A/G beads were used to rule out any non-specific adsorption to the beads. Lanes 1 and 2 of Figure 2C are input controls showing the presence of β2M (lower panel) and ESAT-6 (Lane 1, upper panel) or ESAT-6ΔC (Lane 2, upper panel) in the reaction mixture containing THP-1 lysate and His-tagged ESAT-6 or 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)

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.
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ppat-1004446-g002: 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.
Mentions: The extreme C-terminus of ESAT-6 is a floppy, structurally ill defined region, away from the helical core and is not known to be involved in the interaction with CFP-10 [14]. Mutant M. tuberculosis expressing a truncated ESAT-6 having residues deleted from the C-terminal end was found to be attenuated. However, these mutant strains had a functional ESX-1 system with normal secretion of the ESAT-6:CFP-10 complex [13], [15] indicating that the residues at the C-terminal end of ESAT-6 are not required for a structural role in the formation of a heterodimer with CFP-10 but crucial for the virulence functions of ESAT-6. Therefore, we speculated that the free C-terminus of ESAT-6 is probably involved in the interaction with β2M and such an interaction is possibly critical for M. tuberculosis virulence [21]. Once again using the yeast two hybrid assay, we observed that deletion of the last 6 amino acids (valine, threonine, glycine, methionine, phenylalanine and alanine) from the C-terminal end of ESAT-6 (Figure S2) was sufficient to prevent the interaction of ESAT-6 and β2M (Figure 2A). To further validate the yeast two hybrid data, we used a C-terminally truncated ESAT-6 (ESAT-6ΔC) to carry out GST pull down assay. The GST-tagged full length ESAT-6 protein (ESAT-6-GST) as well as the GST-tagged ESAT-6ΔC (ESAT-6ΔC-GST) protein were incubated with THP-1 extract and the pulled down fractions were probed for the presence of β2M using anti-β2M Ab. It was found that only the full length ESAT-6 protein was able to interact with β2M (Figure 2B). To further confirm our observations, we carried out a Co-IP assay where THP-1 lysates were incubated with full length His-tagged ESAT-6 or His-tagged ESAT-6ΔC and β2M-complexes were immunoprecipitated using anti-β2M Ab. The immunoprecipitated complexes were resolved on SDS-PAGE and probed with either anti-His Ab to detect ESAT-6/ESAT-6ΔC (Figure 2C, upper panel) or anti-β2M Ab to detect β2M (Figure 2C, lower panel). The data shown in Figure 2C clearly demonstrate that anti-β2M antibody can immunoprecipitate the full length ESAT-6 (Figure 2C, Lane 3), but not the mutant ESAT-6ΔC (Figure 2C, Lane 4), indicating that the C-terminal (90–95) residues of ESAT-6 are important for its interaction with β2M. Lane 5 of Figure 2C is negative control where Protein A/G beads were used to rule out any non-specific adsorption to the beads. Lanes 1 and 2 of Figure 2C are input controls showing the presence of β2M (lower panel) and ESAT-6 (Lane 1, upper panel) or ESAT-6ΔC (Lane 2, upper panel) in the reaction mixture containing THP-1 lysate and His-tagged ESAT-6 or 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