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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 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.
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ppat-1004446-g001: 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.

Mentions: We carried out yeast two-hybrid (Y2H) screening to identify the probable ESAT-6 interacting proteins from the host. ESAT-6 cloned in the bait vector pGBKT7 was used to screen a human leukocyte cDNA library cloned into the prey vector pACT2. The suitability of ESAT-6 for use as bait was confirmed by measuring its expression as GAL4-ESAT-6 fusion protein and also by assessing the toxicity of the fusion protein as well as auto-activation of interaction markers. Yeast strain AH109 expressing the Gal4-ESAT-6 fusion protein had normal growth kinetics and auto-activation of the reporter genes was not observed in these transformants. For Y2H screening, Mat-a strain AH109 harboring the bait vector pGBKT7-ESAT-6 was mated with Mat-α strain Y187 transformed with prey library plasmid and the mating mixture was plated on QDO plates (SD/–Ade/–His/–Leu/–Trp) for high stringency of selection. The prey plasmids, rescued from the colonies that appeared on selection plates, were sequenced using 3′ AD Sequencing Primer and were identified by querying these sequences against the NCBI GenBank database using the MegaBlast program. One of these cDNA sequences in the prey plasmid was found to have very high similarity with human beta-2-microglobulin (Figure S1). The interaction was found to be truly positive as the prey plasmid carrying the β2M cDNA sequence (pACT2-β2M) failed to grow in the presence of empty bait vector pGBKT7 alone (Figure 1A).


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 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.
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Related In: Results  -  Collection

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ppat-1004446-g001: 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.
Mentions: We carried out yeast two-hybrid (Y2H) screening to identify the probable ESAT-6 interacting proteins from the host. ESAT-6 cloned in the bait vector pGBKT7 was used to screen a human leukocyte cDNA library cloned into the prey vector pACT2. The suitability of ESAT-6 for use as bait was confirmed by measuring its expression as GAL4-ESAT-6 fusion protein and also by assessing the toxicity of the fusion protein as well as auto-activation of interaction markers. Yeast strain AH109 expressing the Gal4-ESAT-6 fusion protein had normal growth kinetics and auto-activation of the reporter genes was not observed in these transformants. For Y2H screening, Mat-a strain AH109 harboring the bait vector pGBKT7-ESAT-6 was mated with Mat-α strain Y187 transformed with prey library plasmid and the mating mixture was plated on QDO plates (SD/–Ade/–His/–Leu/–Trp) for high stringency of selection. The prey plasmids, rescued from the colonies that appeared on selection plates, were sequenced using 3′ AD Sequencing Primer and were identified by querying these sequences against the NCBI GenBank database using the MegaBlast program. One of these cDNA sequences in the prey plasmid was found to have very high similarity with human beta-2-microglobulin (Figure S1). The interaction was found to be truly positive as the prey plasmid carrying the β2M cDNA sequence (pACT2-β2M) failed to grow in the presence of empty bait vector pGBKT7 alone (Figure 1A).

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