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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

Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER.PMA-differentiated THP-1 macrophages were incubated with FITC-labelled ESAT-6:CFP-10 (green) either at 4°C (A) or 37°C (B) for about 120 minutes. Cells were then washed, fixed, permeabilised and stained for an ER marker calnexin using rabbit anti-calnexin Ab and Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red). In another set of experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal macrophages (E) were treated with FITC-labelled ESAT-6 or ESAT-6:CFP-10 (green) for about 100 minutes at 37°C and then incubated with ER-Tracker dye (blue) for another 20 minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different experiments.
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ppat-1004446-g004: Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER.PMA-differentiated THP-1 macrophages were incubated with FITC-labelled ESAT-6:CFP-10 (green) either at 4°C (A) or 37°C (B) for about 120 minutes. Cells were then washed, fixed, permeabilised and stained for an ER marker calnexin using rabbit anti-calnexin Ab and Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red). In another set of experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal macrophages (E) were treated with FITC-labelled ESAT-6 or ESAT-6:CFP-10 (green) for about 100 minutes at 37°C and then incubated with ER-Tracker dye (blue) for another 20 minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different experiments.

Mentions: Interestingly, ESAT-6 and/or ESAT-6:CFP-10 complex secreted by the M. tuberculosis bacilli in the phagosome or cytoplasm can make its way out by lysing the cell membranes [25], [26] which can be taken up by the surrounding macrophages and dendritic cells. Another possible source of soluble ESAT-6 and/or ESAT-6:CFP-10 is mycobacteria surviving in the extracellular milieu of caseating granulomas where the proteins secreted by the extracellular bacilli are available for uptake by antigen presenting cells present in these granulomas [27]. Interestingly, studies have indicated that soluble proteins can gain direct access to the ER lumen of dendritic cells [28]. Therefore, soluble ESAT-6 and/or ESAT-6:CFP-10 taken up by macrophages and dendritic cells may be retrotranslocated directly to the ER of these cells which would enable it to interact with β2M and modulate β2M-dependent responses. To test this, THP-1 macrophages were incubated for 2 hours with FITC-labelled ESAT-6 or ESAT-6:CFP-10 and their localization was tracked along with the ER-specific marker calnexin by confocal microscopy. The ESAT-6:CFP-10 was found to be present in the ER (Figure 4B). Similarly, FITC-labelled ESAT-6:CFP-10 complex was also found to be present within the ER-tracker dye-positive regions in THP-1 macrophages (Figure 4D) as well as in mouse peritoneal macrophages (Figure 4E). Similarly, FITC-labelled ESAT-6 alone was found to be present in the ER-tracker dye-positive regions of KG-1 dendritic like cells (Figure 4C). FITC-labelled ESAT-6:CFP-10 was incubated with THP-1 macrophages at 4°C as a negative control for internalisation (Figure 4A). These observations suggest that soluble ESAT-6 and ESAT-6:CFP-10 can translocate into the ER lumen from the extracellular milieu to interact with the resident β2M in KG-1 cells and macrophages. From the confocal microscopy data, it appears that ESAT-6:CFP-10 is able to enter the vesicles of the phagocytic pathway as well as other cellular compartments since all the FITC signal emanating from labelled ESAT-6:CFP-10 did not superimpose with the signal emanating from ER-specific markers (Figure 4). However, as is evident from the data, sufficient quantities of ESAT-6/ESAT-6:CFP-10 are able to translocate to the ER where the complex is available for possible interaction with β2M.


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)

Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER.PMA-differentiated THP-1 macrophages were incubated with FITC-labelled ESAT-6:CFP-10 (green) either at 4°C (A) or 37°C (B) for about 120 minutes. Cells were then washed, fixed, permeabilised and stained for an ER marker calnexin using rabbit anti-calnexin Ab and Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red). In another set of experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal macrophages (E) were treated with FITC-labelled ESAT-6 or ESAT-6:CFP-10 (green) for about 100 minutes at 37°C and then incubated with ER-Tracker dye (blue) for another 20 minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004446-g004: Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER.PMA-differentiated THP-1 macrophages were incubated with FITC-labelled ESAT-6:CFP-10 (green) either at 4°C (A) or 37°C (B) for about 120 minutes. Cells were then washed, fixed, permeabilised and stained for an ER marker calnexin using rabbit anti-calnexin Ab and Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red). In another set of experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal macrophages (E) were treated with FITC-labelled ESAT-6 or ESAT-6:CFP-10 (green) for about 100 minutes at 37°C and then incubated with ER-Tracker dye (blue) for another 20 minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different experiments.
Mentions: Interestingly, ESAT-6 and/or ESAT-6:CFP-10 complex secreted by the M. tuberculosis bacilli in the phagosome or cytoplasm can make its way out by lysing the cell membranes [25], [26] which can be taken up by the surrounding macrophages and dendritic cells. Another possible source of soluble ESAT-6 and/or ESAT-6:CFP-10 is mycobacteria surviving in the extracellular milieu of caseating granulomas where the proteins secreted by the extracellular bacilli are available for uptake by antigen presenting cells present in these granulomas [27]. Interestingly, studies have indicated that soluble proteins can gain direct access to the ER lumen of dendritic cells [28]. Therefore, soluble ESAT-6 and/or ESAT-6:CFP-10 taken up by macrophages and dendritic cells may be retrotranslocated directly to the ER of these cells which would enable it to interact with β2M and modulate β2M-dependent responses. To test this, THP-1 macrophages were incubated for 2 hours with FITC-labelled ESAT-6 or ESAT-6:CFP-10 and their localization was tracked along with the ER-specific marker calnexin by confocal microscopy. The ESAT-6:CFP-10 was found to be present in the ER (Figure 4B). Similarly, FITC-labelled ESAT-6:CFP-10 complex was also found to be present within the ER-tracker dye-positive regions in THP-1 macrophages (Figure 4D) as well as in mouse peritoneal macrophages (Figure 4E). Similarly, FITC-labelled ESAT-6 alone was found to be present in the ER-tracker dye-positive regions of KG-1 dendritic like cells (Figure 4C). FITC-labelled ESAT-6:CFP-10 was incubated with THP-1 macrophages at 4°C as a negative control for internalisation (Figure 4A). These observations suggest that soluble ESAT-6 and ESAT-6:CFP-10 can translocate into the ER lumen from the extracellular milieu to interact with the resident β2M in KG-1 cells and macrophages. From the confocal microscopy data, it appears that ESAT-6:CFP-10 is able to enter the vesicles of the phagocytic pathway as well as other cellular compartments since all the FITC signal emanating from labelled ESAT-6:CFP-10 did not superimpose with the signal emanating from ER-specific markers (Figure 4). However, as is evident from the data, sufficient quantities of ESAT-6/ESAT-6:CFP-10 are able to translocate to the ER where the complex is available for possible interaction with β2M.

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