Although nucleotides are critical signaling molecules in all domains of life, cyclic dinucleotides appear to be produced solely by bacteria and archaea. For example, c-di-GMP is a ubiquitous second messenger that regulates biofilm formation, motility, and virulence in diverse bacterial species8
. Recently, cyclic diadenylate monophosphate (c-di-AMP) was discovered as a bacterial regulatory molecule14
, although its role remains to be fully characterized. Since they are unique to microbes, cyclic dinucleotides serve as appropriate targets for immune recognition15
. Indeed, induction of IFN by Listeria monocytogenes
depends on bacterial secretion of cyclic-di-AMP12
. However, it remains unclear how cyclic dinucleotides are sensed in mammalian cells.
To address the mechanism by which mammalian cells sense cyclic dinucleotides, we first confirmed that cyclic dinucleotides are detected in the host cell cytosol10
by expressing RocR, a c-di-GMP-specific phosphodiesterase from Pseudomonas aeruginosa
, in the cytosol of macrophages. In these cells, IFN induction by c-di-GMP (but not other stimuli) is reduced 10-fold compared to vector transduced cells (), confirming that the cytosolic presence of c-di-GMP is important to induce IFN.
STING is sufficient to restore responsiveness to cyclic dinucleotides
To identify candidate cyclic dinucleotide sensors, we sought to identify molecules that could reconstitute the IFN response to cyclic dinucleotides in HEK293T cells, which do not respond to c-di-GMP10
. Since STING is essential for the IFN response to cyclic dinucleotides11
, and STING expression is low/undetectable in HEK293T cells (Supplementary Fig. 1
, data not shown), we first expressed STING in HEK293T cells. Overexpression of STING spontaneously induces an IFN reporter3,6,7
, so we transfected a low amount of STING that by itself was insufficient to induce IFN. To our surprise, low levels of STING were sufficient to reconstitute responsiveness of 293T cells to c-di-GMP () and c-di-AMP (). By contrast, the non-functional goldenticket
) allele of STING (I199N)11
did not restore responsiveness to c-di-GMP (). Interestingly, expression of wild-type STING did not confer responsiveness of 293T cells to double-stranded (ds) DNA oligonucleotides (e.g., vaccinia virus (VV) 70mer or interferon stimulatory DNA (ISD)) that were previously shown to induce type I IFN in macrophages via STING4,16
(, Supplementary Fig. 2a
). By contrast, induction of IFN by poly(dAT:dTA) DNA was identical in cells transfected with wild-type or gt Sting
, demonstrating that the Pol III DNA-sensing pathway17,18
is intact in these cells and is not responsible for detection of c-di-GMP (). As a positive control, Myd88−/−Trif−/−
immortalized macrophages, which express STING, responded similarly to c-di-GMP, poly(dAT:dTA), VV 70mer and ISD (, Supplementary Fig. 2b
). Our results demonstrate STING expression is sufficient to restore responsiveness of HEK293T cells to cyclic dinucleotides but not to DNA.
We next tested whether STING, or perhaps another protein in HEK293T cells, binds to c-di-GMP. We used an in-vitro
ultraviolet (UV) crosslinking assay to identify putative sensor protein(s) in HEK293T cell lysates that interact directly with radiolabelled c-di-GMP (c-di-GMP32)
. We expected to identify directly interacting proteins since only molecules within bond length proximity are efficiently crosslinked by UV19
. We detected a prominent ~40 kDa radiolabelled protein, corresponding to the predicted molecular weight of monomeric STING, in lysates of cells transfected with STING-HA, but not in lysates of cells transfected with STING-HA I199N or vector only (). The ~40 kDa band did not appear when the same lysates were crosslinked with GTP32
, implying that crosslinking to c-di-GMP32
was specific (). We also observed an ~80 kDa species that possibly corresponds to a previously reported STING dimer6
(). To test the hypothesis that STING crosslinks with c-di-GMP32
, we immunoprecipitated STING from transfected HEK293T cells, and performed the c-di-GMP32
binding assay on the immunoprecipitate. Bands corresponding to the molecular weight of STING monomer and dimer were identified only in immunoprecipitates of lysates overexpressing STING and not in mock immunoprecipitates of lysates of vector-transfected cells (). Thus, STING appears to bind c-di-GMP.
STING binds cyclic dinucleotides
To confirm that binding of c-di-GMP32 to STING is specific, we performed the c-di-GMP binding assay in the presence of unlabelled nucleotides. Unlabelled c-di-GMP and c-di-AMP specifically competed with c-di-GMP32 for binding to STING (). By contrast, GTP, other guanosine derivatives, or nucleic acids (including dsDNA), competed away non-specific binding (asterisks, ), but, under our specific assay conditions, could not compete efficiently with c-di-GMP32 for binding to STING (arrows, ). Since the cell cytosol contains high (0.1–1mM) concentrations of GTP, a putative c-di-GMP sensor must exhibit a high degree of specificity for c-di-GMP over GTP. We found that c-di-GMP efficiently crosslinked to STING even in the presence of 1mM GTP ().
Although these data imply that STING directly and specifically binds cyclic dinucleotides, they do not address whether other host proteins might also be required. STING is predicted to encode an N-terminal domain with multiple transmembrane segments, followed by a globular C-terminal domain (CTD). Since the CTD contains the amino acid substitution (I199N) that abolishes STING function in goldenticket
, we hypothesized that the CTD might be involved in binding cyclic dinucleotides. Thus, we subjected purified recombinant His6
-tagged CTD of STING (amino acids 138–378) () to the c-di-GMP32
binding assay. We found that the recombinant CTD of STING bound c-di-GMP32
, and that binding was specifically competed with cold c-di-GMP or c-di-AMP but not cold GTP or ATP (). We used equilibrium dialysis to obtain an estimate of ~5μM for the affinity (Kd
) of c-di-GMP binding to the STING CTD (). In its native membrane-bound form, or in complex with other host factors, STING may exhibit a stronger affinity for c-di-GMP; nevertheless, a 5μM affinity is consistent with the dose response previously observed in macrophages12
. Consistent with the ability of STING to dimerize6
, the binding data suggest a stoichiometry of one molecule of c-di-GMP per two molecules of STING.
In order to identify amino acids involved in c-di-GMP binding and/or IFN induction, we introduced point mutations into STING. Focusing on clusters of conserved and charged residues, we mutated a total of 67 amino acids, individually or in groups, and identified mutants that fell in one of five categories (, Supplementary Table 1
, Supplementary Fig. 3–4
). Class I consists of mutations that abolish both binding and IFN induction (, red, Supplementary Table 1
). Class II mutants bind c-di-GMP but fail to induce IFN (, purple). Class III comprises “hyperactive” mutants that spontaneously induce IFN at low levels of transfection (, green, Supplementary Table 1
). Class IV mutants induced IFN when overexpressed, but were not inducible in response to c-di-GMP (, blue, Supplementary Table 1
). Class V consists of mutants that had no effect on binding or IFN induction (, yellow, Supplementary Table 1
). Although mutation of STING can result in diverse phenotypes, a key finding is that all mutants that failed to bind c-di-GMP also lost the ability to induce IFN in response to c-di-GMP. Consistent with our observation that the CTD is sufficient to bind c-di-GMP (), all mutations that affected c-di-GMP binding were located within the CTD.
DNA and cyclic dinucleotides induce indistinguishable transcriptional responses in macrophages10
and STING appears essential for both responses4,11
. However, in contrast to cyclic dinucleotides, we found STING expression is insufficient to restore responsiveness of HEK293T cells to DNA (). Moreover, our competition assays indicate that DNA does not compete with cyclic-di-GMP for binding to STING under the conditions tested (). Thus, while our data indicate STING functions as a direct immunosensor of cyclic dinucleotides, additional host proteins appear likely to be involved in IFN induction by DNA. Indeed, two candidate DNA sensors, DAI and IFI16, have been identified16,20
, neither of which appear to be essential for the response to cyclic dinucleotides (10
; unpublished data). To determine if responsiveness to cyclic dinucleotides and DNA are separable functions of STING, we sought to identify STING mutants that fail to respond to cyclic dinucleotides but still respond to DNA. We identified a STING mutant (R231A) that was unresponsive to c-di-GMP (), though it still induced IFN when overexpressed () and still bound c-di-GMP (). Interestingly, STING R231A was able to restore responsiveness of goldenticket
bone marrow macrophages to DNA, but not to cyclic-di-GMP (). Thus, cyclic dinucleotide sensing and DNA sensing can be uncoupled, suggesting that these two pathways are discrete but share STING as a common signaling molecule. It is unexpected that STING would function both as a direct immunosensor (of cyclic dinucleotides) and as a signaling adaptor (in the response to DNA). One possibility is that STING initially evolved as a cyclic dinucleotide sensor and was subsequently coopted for DNA sensing.
The IFN response to DNA and c-di-GMP can be uncoupled
We previously used mouse mutagenesis to identify STING as an essential molecule in the in vivo
IFN response to cyclic dinucleotides11
. The requirement for STING can now be rationalized by our proposal that STING functions as a direct sensor of cyclic dinucleotides. Interestingly, STING does not share homology with any known immunosensor, and therefore appears to represent a novel category of microbial detector. Although a BLAST search of the mouse proteome for homologs of the Listeria
diadenylate cyclase (lmo2120; DacA) identifies STING as the top hit, the homology is limited to a short region of the STING CTD (amino acids 311–358). STING does not appear to exhibit homology to PilZ-domain proteins that function as c-di-GMP receptors in bacteria8
. Structural studies are required to determine if STING resembles any known proteins in mammals or bacteria.
Numerous studies have demonstrated that cyclic dinucleotides are potent immunostimulatory compounds that may be valuable as novel immunotherapeutics or adjuvants9,13
. Therapeutic development of cyclic dinucleotides will be greatly facilitated by an improved understanding of the mechanism by which they are sensed. Furthermore, our finding that STING is a direct detector of cyclic dinucleotides provides insight into the fundamental mechanisms by which the innate immune system can detect bacterial infection.