Bacterial cell volume regulation and the importance of cyclic di-AMP.

SUMMARYNucleotide-derived second messengers are present in all domains of life. In prokaryotes, most of their functionality is associated with general lifestyle and metabolic adaptations, often in response to environmental fluctuations of physical parameters. In the last two decades, cyclic di-AMP has emerged as an important signaling nucleotide in many prokaryotic lineages, including Firmicutes, Actinobacteria, and Cyanobacteria. Its importance is highlighted by the fact that both the lack and overproduction of cyclic di-AMP affect viability of prokaryotes that utilize cyclic di-AMP, and that it generates a strong innate immune response in eukaryotes. In bacteria that produce the second messenger, most molecular targets of cyclic di-AMP are associated with cell volume control. Besides, other evidence links the second messenger to cell wall remodeling, DNA damage repair, sporulation, central metabolism, and the regulation of glycogen turnover. In this review, we take a biochemical, quantitative approach to address the main cellular processes that are directly regulated by cyclic di-AMP and show that these processes are very connected and require regulation of a similar set of proteins to which cyclic di-AMP binds. Altogether, we argue that cyclic di-AMP is a master regulator of cell volume and that other cellular processes can be connected with cyclic di-AMP through this core function. We further highlight important directions in which the cyclic di-AMP field has to develop to gain a full understanding of the cyclic di-AMP signaling network and why some processes are regulated, while others are not.

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Synthesis, kinetic studies, and QSAR of dinucleoside polyphosphate derivatives as human AK1 inhibitors.

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.

Allosteric regulation by c-di-AMP modulates a complete N-acetylglucosamine signaling cascade in Saccharopolyspora erythraea.

c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.

A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA.

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

c-di-AMP determines the hierarchical organization of bacterial RCK proteins.

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

Bacterial second messenger cyclic di-AMP in streptococci.

Cyclic dimeric adenosine monophosphate (c-di-AMP) has been well studied in bacteria, including those of the genus Streptococcus, since the first recognition of this dinucleotide in 2008. Streptococci possess a sole diadenylate cyclase, CdaA, and distinct c-di-AMP phosphodiesterases. Interestingly, cdaA is required for viability of some streptococcal species but not all when streptococci are grown in standard laboratory media. Bacteria of this genus also have distinct c-di-AMP effector proteins, diverse c-di-AMP-signaling pathways, and subsequent biological outcomes. In streptococci, c-di-AMP may influence bacterial growth, morphology, biofilm formation, competence program, drug resistance, and bacterial pathogenesis. c-di-AMP secreted by streptococci has also been shown to interact with the mammalian host and induces immune responses including type I interferon production. In this review, we summarize the reported c-di-AMP networks in seven species of the genus Streptococcus, which cause diverse clinical manifestations, and propose future perspectives to investigate the signaling molecule in these streptococcal pathogens.

Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase.

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.

Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation.

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

All DACs in a Row: Domain Architectures of Bacterial and Archaeal Diadenylate Cyclases.

Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain dac genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.

Bacteriophage antidefense genes that neutralize TIR and STING immune responses.

Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.

The c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Regulatory mechanisms of c-di-AMP synthase from Mycobacterium smegmatis revealed by a structure: Function analysis.

Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue.

The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation.

Diadenosine polyphosphates (Apn As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Apn As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap4 A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4 A levels during cellular stress differs from when Ap4 A is present at low concentrations, allowing response to extracellular cues.

Chemical proteomics reveals interactors of the alarmone diadenosine triphosphate in the cancer cell line H1299.

Intracellular dinucleoside polyphosphates (Npn Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap3 A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap3 A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap3 A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap3 A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap3 A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit.

Recognition of cyclic dinucleotides and folates by human SLC19A1.

Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A122,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.

Development of Nucleoside Diphosphate-Bearing Fragile Histidine Triad-Imaging Fluorescence Probes with Well-Tuned Hydrophobicity for Intracellular Delivery.

Fluorescence-guided cancer surgery can dramatically improve recurrence rates and postoperative quality of life of patients by accurately distinguishing the boundary between normal and cancer tissues during surgery, thereby minimizing excision of normal tissue. One promising target in early stage cancer is fragile histidine triad (FHIT), a cancer suppressor protein with dinucleoside triphosphate hydrolase activity. In this study, we have developed fluorescence probes containing a nucleoside diphosphate moiety, which dramatically improves the reactivity and specificity for FHIT, and a moderately lipophilic ester moiety to increase the membrane permeability. The ester moiety is cleaved by ubiquitous intracellular esterases, and then, FHIT in the cells specifically cleaves nucleoside monophosphate. The remaining phosphate moiety is rapidly cleaved by ubiquitous intracellular phosphatases to release the fluorescent dye. We confirmed that this probe can detect FHIT activity in living cells. A comprehensive evaluation of the effects of various ester moieties revealed that probes with CLogP = 5-7 showed good membrane permeability and were good substrates of the target enzyme; these findings may be helpful in the rational design of other multiple phosphate-containing probes targeting intracellular enzymes.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence.

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3',5')-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe-microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

Cryo-EM structure of an active bacterial TIR-STING filament complex.

Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1-4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4-13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.