bis(3--5-)-cyclic-diguanylic-acid and cyclic-guanosine-monophosphate-adenosine-monophosphate

The regulation of multiple bacterial phenotypes was found to depend on different cyclic dinucleotides (CDNs) that constitute intracellular signaling second messenger systems. Most notably, c-di-GMP, along with proteins related to its synthesis, sensing, and degradation, was identified as playing a central role in the switching from biofilm to planktonic modes of growth. Recently, this research topic has been under expansion, with the discoveries of new CDNs, novel classes of CDN receptors, and the numerous functions regulated by these molecules. In this review, we comprehensively describe the three main bacterial enzymes involved in the synthesis of c-di-GMP, c-di-AMP, and cGAMP focusing on description of their three-dimensional structures and their structural similarities with other protein families, as well as the essential residues for catalysis. The diversity of CDN receptors is described in detail along with the residues important for the interaction with the ligand. Interestingly, genomic data strongly suggest that there is a tendency for bacterial cells to use both c-di-AMP and c-di-GMP signaling networks simultaneously, raising the question of whether there is crosstalk between different signaling systems. In summary, the large amount of sequence and structural data available allows a broad view of the complexity and the importance of these CDNs in the regulation of different bacterial behaviors. Nevertheless, how cells coordinate the different CDN signaling networks to ensure adaptation to changing environmental conditions is still open for much further exploration.

Topics: Bacteria; Bacterial Proteins; Binding Sites; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Models, Molecular; Nucleotides, Cyclic; Plankton; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Signal Transduction

Cyclic dinucleotides (CDNs) are highly versatile signalling molecules that control various important biological processes in bacteria. The best-studied example is cyclic di-GMP (c-di-GMP). Known since the late 1980s, it is now recognized as a near-ubiquitous second messenger that coordinates diverse aspects of bacterial growth and behaviour, including motility, virulence, biofilm formation and cell cycle progression. In this Review, we discuss important new insights that have been gained into the molecular principles of c-di-GMP synthesis and degradation, which are mediated by diguanylate cyclases and c-di-GMP-specific phosphodiesterases, respectively, and the cellular functions that are exerted by c-di-GMP-binding effectors and their diverse targets. Finally, we provide a short overview of the signalling versatility of other CDNs, including c-di-AMP and cGMP-AMP (cGAMP).

Topics: Bacteria; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Nucleotides, Cyclic; Second Messenger Systems; Signal Transduction

Cyclic (c-di-GMP) is the prevalent intracellular signaling intermediate in bacteria. It triggers a spectrum of responses that cause bacteria to shift from a swarming motile phase to sessile biofilm formation. However, additional functions for c-di-GMP and roles for related molecules, such as c-di-AMP and c-AMP-GMP continue to be uncovered. The first usage of cyclic-di-nucleotide (c-di-NMP) signaling in the eukaryote domain emerged only recently. In dictyostelid social amoebas, c-di-GMP is a secreted signal that induces motile amoebas to differentiate into sessile stalk cells. In humans, c-di-NMPs, which are either produced endogenously in response to foreign DNA or by invading bacterial pathogens, trigger the innate immune system by activating the expression of interferon genes. STING, the human c-di-NMP receptor, is conserved throughout metazoa and their closest unicellular relatives, suggesting protist origins for human c-di-NMP signaling. Compared to the limited number of conserved protein domains that detect the second messengers cAMP and cGMP, the domains that detect the c-di-NMPs are surprisingly varied.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Dictyostelium; Dinucleoside Phosphates; Humans; Immunity, Innate; Membrane Proteins; Nucleotides, Cyclic; Nucleotidyltransferases; Phylogeny; Protein Structure, Tertiary; Second Messenger Systems; Signal Transduction

Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2'3' cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3'3' cGAMP, 2'3' cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings. Graphical abstract.

Topics: Animals; Chromatography, Liquid; Cyclic GMP; Dinucleoside Phosphates; Escherichia coli; Fundulidae; Nucleotides, Cyclic; Tandem Mass Spectrometry

Myxococcus xanthus has a nutrient-regulated biphasic life cycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger 3'-5', 3'-5 cyclic di-GMP (c-di-GMP) is essential during both stages of the life cycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct life cycle stages. To address this stage specificity, we determined transcript levels using transcriptome sequencing (RNA-seq) and transcription start sites using Cappable sequencing (Cappable-seq) during growth and development genome wide. All 70 genes encoding c-di-GMP-associated proteins were expressed, with 28 upregulated and 10 downregulated during development. Specifically, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage specifically. By combining operon mapping with published chromatin immunoprecipitation sequencing (ChIP-seq) data for MrpC (M. Robinson, B. Son, D. Kroos, L. Kroos, BMC Genomics 15:1123, 2014, http://dx.doi.org/10.1186/1471-2164-15-1123), the cAMP receptor protein (CRP)-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses the expression of

Topics: Bacterial Proteins; Cyclic AMP Receptor Protein; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Myxococcus xanthus; Nucleotides; Phosphoric Diester Hydrolases

Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αβ (IFN-αβ), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αβ in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy.

Topics: Animals; Cyclic GMP; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; Drug Synergism; Female; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Insulin; Membrane Proteins; Mice; Mice, Inbred NOD; Nanoparticles; Nucleotides, Cyclic; Polymorphism, Genetic; T-Lymphocytes; Up-Regulation

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3'3'-cyclic GMP-AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5'-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5'-pGpG-Ca2+ structure, β5-α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5'-pGpG-Ca2+ structure quite different from other 5'-pGpG bound structures reported earlier.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Vibrio cholerae

How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.

Topics: Binding Sites; Cyclic GMP; Dimerization; Endoplasmic Reticulum; HEK293 Cells; Humans; Ligands; Membrane Proteins; Models, Molecular; Mutagenesis, Site-Directed; Nucleotides, Cyclic; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Signal Transduction

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

Topics: Algorithms; Bacterial Proteins; Cyclic GMP; Dinucleoside Phosphates; Kinetics; Ligases; Magnesium; Molecular Structure; Nucleotides, Cyclic; Protein Binding; Vibrio cholerae

In certain structural or functional contexts, RNA structures can contain protonated nucleotides. However, a direct role for stably protonated nucleotides in ligand binding and ligand recognition has not yet been demonstrated unambiguously. Previous X-ray structures of c-GAMP binding riboswitch aptamer domains in complex with their near-cognate ligand c-di-GMP suggest that an adenine of the riboswitch either forms two hydrogen bonds to a G nucleotide of the ligand in the unusual enol tautomeric form or that the adenine in its N1 protonated form binds the G nucleotide of the ligand in its canonical keto tautomeric state. By using NMR spectroscopy we demonstrate that the c-GAMP riboswitches bind c-di-GMP using a stably protonated adenine in the ligand binding pocket. Thereby, we provide novel insights into the putative biological functions of protonated nucleotides in RNA, which in this case influence the ligand selectivity in a riboswitch.

Topics: Adenine; Cyclic GMP; Ligands; Magnetic Resonance Spectroscopy; Nucleotides, Cyclic; Protein Binding; Riboswitch; RNA; RNA, Bacterial; Vibrio cholerae

Riboswtich RNAs can control gene expression through the structural change induced by the corresponding small-molecule ligands. Molecular dynamics simulations and free energy calculations on the aptamer domain of the 3',3'-cGAMP riboswitch in the ligand-free, cognate-bound and noncognate-bound states were performed to investigate the structural features of the 3',3'-cGAMP riboswitch induced by the 3',3'-cGAMP ligand and the specificity of ligand recognition. The results revealed that the aptamer of the 3',3'-cGAMP riboswitch in the ligand-free state has a smaller binding pocket and a relatively compact structure versus that in the 3',3'-cGAMP-bound state. The binding of the 3',3'-cGAMP molecule to the 3',3'-cGAMP riboswitch induces the rotation of P1 helix through the allosteric communication from the binding sites pocket containing the J1/2, J1/3 and J2/3 junction to the P1 helix. Simultaneously, these simulations also revealed that the preferential binding of the 3',3'-cGAMP riboswitch to its cognate ligand, 3',3'-cGAMP, over its noncognate ligand, c-di-GMP and c-di-AMP. The J1/2 junction in the 3',3'-cGAMP riboswitch contributing to the specificity of ligand recognition have also been found.

Topics: Allosteric Regulation; Binding Sites; Cyclic GMP; Hydrogen Bonding; Ligands; Molecular Dynamics Simulation; Nucleic Acid Conformation; Nucleotides, Cyclic; Principal Component Analysis; Riboswitch; Thermodynamics; Time Factors

Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3'3'-cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents.

Topics: Adjuvants, Immunologic; Animals; Cell-Penetrating Peptides; CpG Islands; Cyclic GMP; Cytokines; Humans; Immunity, Innate; Interferon Type I; Membrane Proteins; Mice; Mice, Inbred C57BL; Nucleotides, Cyclic; Oligodeoxyribonucleotides; Peptides; Spleen; Tumor Cells, Cultured