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Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages

Nature Immunology volume 8, pages 610–618 (2007)Cite this article

A Corrigendum to this article was published on 17 February 2015

This article has been updated

Abstract

Expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is associated with lower innate immune responses to infection. Here we show that ESAT-6 inhibited activation of transcription factor NF-κB and interferon-regulatory factors (IRFs) after Toll-like receptor (TLR) signaling; inhibition of TLR signaling by ESAT-6 required the kinase Akt. Direct binding of ESAT-6 to TLR2 activated Akt and prevented interaction between the adaptor MyD88 and 'downstream' kinase IRAK4, thus abrogating NF-κB activation. The six carboxy-terminal amino acid residues of ESAT-6 were required and sufficient for the TLR2-mediated inhibitory effect. A critical function for the carboxy-terminal peptide of ESAT-6 in restricting MyD88-dependent TLR signaling emphasizes the possibility that mimetic inhibitory peptides could be used to restrict innate immune responses in situations in which prolonged TLR signaling has deleterious effects.

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Figure 1: ESAT-6 inhibits LPS-driven production of IL-12p40.
Figure 2: ESAT-6 inhibits IL-12p40 production elicited by TLR2, TLR7 and TLR9 ligands, Il12 promoter–driven luciferase expression and NF-κB activation.
Figure 3: ESAT-6 inhibits the MyD88-IRAK4 interaction, IRAK4 activation and TRAF6 ubiquitination.
Figure 4: ESAT-6 binds to the surfaces of RAW cells in a TLR2-dependent way.
Figure 5: ESAT-6-mediated inhibition of TLR-driven IL-12p40 production and the MyD88-IRAK4 interaction depend on Akt.
Figure 6: Inhibition by ESAT-6 of MyD88-mediated TLR signaling in BMDMs requires TLR2 and Akt.
Figure 7: ESAT-6 modulates the nuclear translocation of IRF5 and IRF1.
Figure 8: ESAT-6 inhibits the activation of ASK1 and p38 and induction of Ifnb.
Figure 9: The C terminus of ESAT-6 mediates its inhibitory effects on TLR signaling.

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  • 20 November 2014

    In the version of this article initially published, lanes 1, 2 and 4 of Figure 5b included duplicates. The correct immunoblot is now presented. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The Il12b-driven luciferase reporter was from S. Smale (University of California, Los Angeles); 4× NF-κB–luc was from A. Brent Carter (University of Iowa); hemagglutinin-tagged TLR1 and TLR6 were from A. Hajjar (University of Washington School of Medicine, Seattle); hemagglutinin-tagged ubiquitin was from P. Howley (Harvard Medical School); hemagglutinin-tagged IRF3 and IRF5 were from T. Taniguchi (University of Tokyo); and TLR2-knockout mice were from S. Akira (Osaka University). Supported by the Council of Scientific and Industrial Research of the Government of India (S.K.P.) and the Department of Biotechnology of the Government of India.

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  1. Asima Bhattacharyya

    Present address: Present address: Department of Internal Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.,

Authors and Affiliations

  1. Department of Chemistry, Bose Institute, Kolkata, 700 009, India

    Sushil Kumar Pathak, Sanchita Basu, Kunal Kumar Basu, Anirban Banerjee, Shresh Pathak, Asima Bhattacharyya, Manikuntala Kundu & Joyoti Basu

  2. Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology, Tsurumi-ku, Yokohama City, 230-0045, Kanagawa, Japan

    Tsuneyasu Kaisho

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Contributions

S.K.P. did research and analyzed data; S.B., K.B., A. Banerjee, S.P. and A. Bhattacharyya did research; T.K. contributed tools; and M.K. and J.B. designed the research, analyzed data and wrote the paper.

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Supplementary information

Supplementary Fig. 1

ESAT-6-dependent effects on RAW264.7 macrophages are not due to IL-10 induction and ESAT-6 does not modulate TLR ligand-dependent TLR-MyD88 interactions. (PDF 119 kb)

Supplementary Fig. 2

ESAT-6 inhibits the interaction of MyD88 with IRAK-4. (PDF 98 kb)

Supplementary Fig. 3

ESAT-6 does not induce MyD88s or IRAK-M, nor does it alter expression of TLRs and negative regulators of TLR signaling. (PDF 108 kb)

Supplementary Fig. 4

ESAT-6-mediated attenuation of IL-12 p40 release does not require TLR1, TLR6 or CD14 and ESAT-6 attenuates TLR ligand-induced IRAK-4 kinase activity in a PI-3K/Akt-dependent manner. (PDF 127 kb)

Supplementary Fig. 5

ESAT-6 interacts directely with the extracellular domain (ECD) of TLR2. (PDF 37 kb)

Supplementary Fig. 6

ESAT-6 inhibits TLR ligand-induced activation of IKKβ kinase activity, LPS-driven activation of p38 MAPK and nuclear translocation of IRF-3 in RAW cells. (PDF 154 kb)

Supplementary Fig. 7

The C terminus of ESAT-6 inhibits TLR ligand induced nuclear translocation of IRFs 5, 1 and 3, binds to the surface of RAW cells and inhibits TLR ligand-induced IL12p40 production in bone marrow derived macrophages (BMDMs). (PDF 131 kb)

Supplementary Table 1

Sequences of overlapping peptides of ESAT-6. (PDF 8 kb)

Supplementary Table 2

Primers for RT-PCR. (PDF 7 kb)

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Pathak, S., Basu, S., Basu, K. et al. Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 8, 610–618 (2007). https://doi.org/10.1038/ni1468

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