Assays for immunology and microbiology research quantify mammalian and bacterial CDNs

Cyclic dinucleotides (CDNs) serve signaling functions in all domains of life. In eukaryotes, CDN signaling systems provide a means to sense and respond to pathogen infection by detecting foreign nucleic acid species. In prokaryotes, CDNs target multiple effectors to control diverse processes important for wide-ranging biological functions. CDN-based signaling in bacterial cells also has an immunological function that activates a cellular suicide program to protect the bacterial community through abortive phage infection. When an infectious insult is sensed, CDNs work as second messengers to relay signals to effector proteins whose ultimate actions eliminate the pathogen.

Synthesis, Composition, and Degradation

CDNs are heterocyclic molecules synthesized by the cyclization of two ribonucleoside triphosphate moieties via 3’5’ (canonical) or 2’5’ (non-canonical) phosphodiester linkages. Prokaryotic 3’3’-CDNs (c[N(3’5’)pN(3’5’)p]) contain two canonical phosphodiester linkages, while eukaryote 2’3’-cyclic GMP-AMP (cGAMP) (c[G(2’5’)pA(3’5’)p]) contains mixed phosphodiester bonds. A conserved family of nucleotidyltransferases, including the dinucleotide cyclase in V. cholerae (DncV) and its metazoan homolog cyclic GMP-AMP synthase (cGAS), synthesize CDN second messengers. To avoid excessive stimulation, CDN signaling is inactivated by phosphodiesterase (PDE) hydrolysis of one or both phosphodiester bonds. The ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), has been identified as the predominate 2’3’-cGAMP PDE. Cyclic di-AMP is hydrolyzed by species-specific PDEs that contain DHH-DHHA1 or HD domains. For example, in L. monocytogenes, PdeA and PgpH act cooperatively to hydrolyze cyclic di-AMP, and several, nonredundant 3’3’-cGAMP-selective PDEs have been identified in V. cholerae.

Structure models of cyclic di-GMP (PDB 2RDE), cyclic di-AMP (PDB 4YXM), 3’3’-cGAMP (PDB 5CFM), and 2’3’-cGAMP (PDB 4KSY). Note that 3’3’-cGAMP adopts two conformations.

Bacterial Lifestyle Regulators

Different classes of CDNs, including di-purines, hybrids of purines and pyrimidines, and cyclic trinucleotides, regulate multiple bacterial functions. The most well-known involve cyclic di-GMP, which coordinates the transition from a mobile single cell undergoing planktonic growth to sessile growth by controlling bacterial motility to form a multicellular community or biofilm. This CDN is also involved in the regulation of adhesion, cell cycle progression, cell division, quorum sensing, and the synthesis and secretion of virulence factors. Cyclic di-AMP regulates processes important for osmoprotection, cell wall homeostasis, potassium ion channel expression and function, DNA repair, gene expression, biofilm formation, sporulation, antibiotic resistance, and metabolism. 3’3’-cGAMP modulates chemotaxis, virulence, and the use of insoluble extracellular terminal electron acceptors. 3’3’-cGAMP, along with cUAMP (cyclic uridine adenosine monophosphate), also activates phospholipase activity. Much is still left to be discovered about how bacteria coordinate different CDNs to regulate their phenotype.

CDN-Based Antiphage Signaling

CDN nucleotidyltransferases are activated following bacteriophage infection, generating 3’3’-CDNs such as cyclic di-AMP and cyclic di-GMP from the NTPs ATP and GTP, respectively. Binding of these CDNs to latent phospholipases or other effectors such as endonucleases, transmembrane proteins, Toll-interleukin receptor-like (TIR) proteins, and targets of unknown function results in the hydrolysis of the bacterial inner cell membrane and subsequent bacteriolysis.

The cyclic oligonucleotide-based antiphage signaling system.

cGAS-STING Innate Immune Response

Eukaryotic cells evolved a strategy to detect some bacterial CDNs, including 3’3’-cGAMP, cyclic di-AMP, and cyclic di-GMP, in addition to eukaryotic 2’3’-cGAMP molecules. Upon sensing foreign DNA from a pathogen or misplaced self-DNA, cGAS is activated in eukaryotes to produce 2’3’-cGAMP. STING senses 2’3’-cGAMP and initiates type I interferon transcriptional responses to foster innate immunity. Once produced, systems are in place to amplify this signaling by transferring 2’3’-cGAMP via gap junctions or virion packages to neighboring cells or secreting 2’3’-cGAMP into the extracellular space to be sensed by patrolling immune cells. Some microbes such as Listeria bypass cGAS and directly activate STING.

The cGAS-cGAMP-STING signaling axis is activated by accumulating pathogenic or mislocalized self-dsDNA species. 

STING recruits the kinase TBK1 to the transcription factor IRF3 ultimately resulting in type I interferon antiviral activation.

STING-Independent Immune Activation

Apart from STING, other target proteins that interact with CDNs have been identified. Cyclic di-GMP and cyclic di-AMP can directly bind DDX41, a cytosolic helicase that, when bound by a CDN, activates STING. 2’3’-cGAMP inhibits the AMPK signaling complex that represses the autophagy regulator ULK1, which triggers the degradation of STING. Cyclic nucleoside monophosphates can inhibit cAMP activation of HCN4, an ion channel that regulates pacemaker currents within the heart. The antibacterial component siderocalin (LCN2) can directly bind cyclic di-GMP rendering it unable to bind bacterial ferric siderophores causing a loss of its antibacterial activity. Future studies will be needed to substantiate the physiological roles of these effectors in CDN signal transduction.

Therapeutic Potential

With the discovery that CDNs serve as potent activators of the innate immune system, they have become a highly investigated and intriguing pharmacological target in microbial infections, cancer immunotherapy, and autoimmune disease. For instance, cGAS-STING signaling within tumor cells can drive an antitumor response, providing a rationale to use CDNs as STING ligands for antitumor therapy. The immunostimulatory properties of CDNs could similarly be harnessed as prophylactic vaccine adjuvants to induce antigen-specific immune responses after vaccination. Sensitive tools to detect and quantify CDNs will be useful in this pursuit.

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Suggested Reading

da Purificação, A.D., de Azevedo, N.M., de Araujo, G.G., et al. The world of cyclic dinucleotides in bacterial behavior. Molecules 25(10), 2462 (2020).

Mankan, A.K., Müller, M., Witte, G., et al. Cyclic dinucleotides in the scope of the mammalian immune system. Handb. Exp. Pharmacol. 238, 269-289 (2017).

Zaver, S.A. and Woodward, J.J. Cyclic dinucleotides at the forefront of innate immunity. Current Opin. Cell Bio. 63, 49-56 (2020).