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STING is a direct innate immune sensor of cyclic di-GMP

Abstract

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production1,2, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1–IRF3 signalling axis3,4,5,6,7. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria8, have also been shown to induce a STING-dependent type I IFN response9,10,11,12. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics9,13, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

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Figure 1: STING is sufficient to restore responsiveness to cyclic dinucleotides.
Figure 2: STING binds cyclic dinucleotides.
Figure 3: Mutational analysis of STING.
Figure 4: The IFN response to DNA and c-di-GMP can be uncoupled.

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Acknowledgements

We thank H. Carlson, K. Collins, S. McWhirter, D. Raulet, K. Sjölander and members of the Vance, Barton and Portnoy laboratories at the University of California, Berkeley, for advice and discussions. We thank J. Woodward and D. Portnoy for their gift of purified c-di-AMP. Work in R.E.V.’s laboratory is supported by investigator awards from the Burroughs Wellcome Fund and the Cancer Research Institute and by National Institutes of Health (NIH) grants AI075039, AI080749 and AI063302. D.L.B. is supported by an NIH National Research Service Award fellowship F32 (AI091100).

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D.L.B. performed luciferase assays and quantitative RT–PCR, generated c-di-[32P]GMP, purified recombinant STING, performed c-di-[32P]GMP binding assays and transduced gt bone marrow macrophages. K.M.M. generated truncation mutations and performed luciferase assays. K.S.-T. generated point mutants and performed luciferase assays. D.L.B., K.M.M. and R.E.V. participated in study design and data analysis. D.L.B. and R.E.V. wrote the paper. B.E. contributed to protein purification methods. J.S.I. contributed to the design of the equilibrium dialysis experiments and the analysis of binding data. M.H. and Y.H. synthesized c-di-GMP.

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Correspondence to Russell E. Vance.

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The authors declare no competing financial interests.

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Burdette, D., Monroe, K., Sotelo-Troha, K. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011). https://doi.org/10.1038/nature10429

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