Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS-cGAMP-STING Pathway.

In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)-2'3'-cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which thecGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases.

Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

SLC19A1 Is an Importer of the Immunotransmitter cGAMP.

2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen, we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

DNA-induced 2'3'-cGAMP enhances haplotype-specific human STING cleavage by dengue protease.

The cytosolic DNA sensor cGMP-AMP synthase (cGAS) synthesizes the noncanonical cyclic dinucleotide 2'3'-cGAMP to activate the adaptor protein stimulator of IFN genes (STING), thus awakening host immunity in response to DNA pathogen infection. However, dengue virus (DENV), an RNA virus without a DNA stage in its life cycle, also manipulates cGAS-STING-mediated innate immunity by proteolytic degradation of STING. Here, we found that the sensitivity of STING to DENV protease varied with different human STING haplotypes. Exogenous DNA further enhanced DENV protease's ability to interact and cleave protease-sensitive STING. DNA-enhanced STING cleavage was reduced in cGAS-knockdown cells and triggered by the cGAS product 2'3'-cGAMP. The source of DNA may not be endogenous mitochondrial DNA but rather exogenous reactivated viral DNA. Cells producing 2'3'-cGAMP by overexpressing cGAS or with DNA virus reactivation enhanced STING cleavage in neighboring cells harboring DENV protease. DENV infection reduced host innate immunity in cells with the protease-sensitive STING haplotype, whose homozygote genotype frequency was found significantly reduced in Taiwanese people with dengue fever. Therefore, the human STING genetic background and DNA pathogen coinfection may be the missing links contributing to DENV pathogenesis.

Brucella abortus Cyclic Dinucleotides Trigger STING-Dependent Unfolded Protein Response That Favors Bacterial Replication.

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-ß, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-ß is also important for the UPR induction during B. abortus infection. Indeed, IFN-ß shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-ß favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-ß signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.

Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae.

Sensing and responding to environmental changes is essential for bacteria to adapt and thrive, and nucleotide-derived second messengers are central signaling systems in this process. The most recently identified bacterial cyclic dinucleotide second messenger, 3', 3'-cyclic GMP-AMP (cGAMP), was first discovered in the El Tor biotype of Vibrio cholerae The cGAMP synthase, DncV, is encoded on the VSP-1 pathogenicity island, which is found in all El Tor isolates that are responsible for the current seventh pandemic of cholera but not in the classical biotype. We determined that unregulated production of DncV inhibits growth in El Tor V. cholerae but has no effect on the classical biotype. This cGAMP-dependent phenotype can be suppressed by null mutations in vc0178 immediately 5' of dncV in VSP-1. VC0178 [renamed as cGAMP-activated phospholipase in Vibrio (CapV)] is predicted to be a patatin-like phospholipase, and coexpression of capV and dncV is sufficient to induce growth inhibition in classical V. cholerae and Escherichia coli Furthermore, cGAMP binds to CapV and directly activates its hydrolase activity in vitro. CapV activated by cGAMP in vivo degrades phospholipids in the cell membrane, releasing 16:1 and 18:1 free fatty acids. Together, we demonstrate that cGAMP activates CapV phospholipase activity to target the cell membrane and suggest that acquisition of this second messenger signaling pathway may contribute to the emergence of the El Tor biotype as the etiological agent behind the seventh cholera pandemic.

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides.

BACKGROUND: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3',5'-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. RESULTS: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. CONCLUSIONS: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.

cCMP and cUMP: emerging second messengers.

The cyclic purine nucleotides cAMP and cGMP are established second messengers. By contrast, the existence of the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) has been controversial for decades. The recent development of highly sensitive mass spectrometry (MS) methods allowed precise quantitation and unequivocal identification of cCMP and cUMP in cells. Importantly, cCMP and cUMP generators, effectors, cleaving enzymes, and transporters have now been identified. Here, I discuss evidence in support of cCMP and cUMP as bona fide second messengers, the emerging therapeutic implications of cCMP and cUMP signaling, and important unresolved questions for this field.

Overlapping Patterns of Rapid Evolution in the Nucleic Acid Sensors cGAS and OAS1 Suggest a Common Mechanism of Pathogen Antagonism and Escape.

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular 'arms races.' Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2'-5'-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP.

Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

Control of bacterial exoelectrogenesis by c-AMP-GMP.

Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-ß during Chlamydia trachomatis infection.

IFN-ß has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-ß expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-ß expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-ß expression. We determined that cGAS is required for IFN-ß expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-ß, coculture of cells depleted for either STING or cGAS rescued IFN-ß expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-ß expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-ß expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

The structural basis for 2'-5'/3'-5'-cGAMP synthesis by cGAS.

cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2'-5'/3'-5'-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.

Evolutionary genomics reveals the premetazoan origin of opposite gating polarity in animal-type voltage-gated ion channels.

Electrical signaling in animals ensures the rapid and accurate transmission of information, often carried by voltage-gated Na(+), Ca(2+) and K(+) channels that are activated by membrane depolarization. In heart and neurons, a distinct type of ion channel called the hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channel is activated by membrane hyperpolarization. Recent genomic studies have revealed that animal-type voltage-gated Na(+) channels (Liebeskind BJ, et al. 2011. Proc Natl Acad Sci U S A. 108:9154) had evolved in choanoflagellates, one of the unicellular relatives of animals. To date, HCN channels have been considered to be animal-specific. Here, we demonstrate the presence of an HCN channel homolog (SroHCN) in the choanoflagellate protist Salpingoeca rosetta. SroHCN contains highly conserved functional domains and sequence motifs that are correlated with the unique biophysical activities of HCN channels. These findings provide novel genomic insights into the evolution of complex electrical signaling before the emergence of multicellular animals.

An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP.

It has been revealed that 2'3'-cyclic-GMP-AMP (cGAMP), a second messenger that activates the antiviral stimulator of IFN genes (STING), elicits an antitumoral immune response. Since cGAMP cannot cross the cell membrane, it is not clear how intracellular STING has been activated by extracellular cGAMP until SLC19A1 was identified as an importer to transport extracellular cGAMP into the cytosol. However, SLC19A1-deficient cells also sense extracellular cGAMP, suggesting the presence of mechanisms other than the facilitating transporters for STING sensing extracellular cGAMP. Here, using immunoprecipitation, immunofluorescence, and flow cytometry, we identified an alternatively spliced STING isoform, plasmatic membrane STING (pmSTING), that localized in the plasma membrane with its C-terminus outside the cell, due to a lack of 1 transmembrane domain in its N-terminus compared with canonical STING. Further studies showed that extracellular cGAMP not only promoted the dimerization of pmSTING and interaction of pmSTING with TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3), but also enhanced the phosphorylation of TBK1 and IRF3 and the production of IFN in pmSTING-transfected cells. Additionally, we also identified similar pmSTING isoforms in other species including human. This study suggests a conserved role for pmSTING in sensing extracellular cGAMP and provides insight into the role of cGAMP as an immunotransmitter.

Design and synthesis of gene-directed caged cyclic nucleotides exhibiting cell type selectivity.

We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.

The STING phase-separator suppresses innate immune signalling.

Biomolecular condensates (biocondensates) formed via liquid-liquid phase-separation of soluble proteins have been studied extensively. However, neither the phase-separation of endoplasmic reticulum (ER) transmembrane protein nor a biocondensate with organized membranous structures has been reported. Here, we have discovered a spherical ER membranous biocondensate with puzzle-like structures caused by condensation of the ER-resident stimulator of interferon genes (STING) in DNA virus-infected or 2'3'-cGAMP (cyclic GMP-AMP)-treated cells, which required STING transmembrane domains, an intrinsically disordered region (IDR) and a dimerization domain. Intracellular 2'3'-cGAMP concentrations determined STING translocation or condensation. STING biocondensates constrained STING and TBK1 (TANK binding protein 1) to prevent innate immunity from overactivation, presumably acting like a 'STING-TBK1-cGAMP sponge'. Cells expressing STING-E336G/E337G showed notably enhanced innate immune responses due to impaired STING condensation after viral infection at later stages. Microtubule inhibitors impeded the STING condensate gel-like transition and augmented type I-interferon production in DNA virus-infected cells. This membranous biocondensate was therefore named the STING phase-separator.