cGLRs are a diverse family of pattern recognition receptors in innate immunity.

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides.

Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells. Surprisingly, the Acb2 hexamer also binds with high affinity to CBASS cyclic trinucleotides (CTNs) 3'3'3'-cyclic AMP-AMP-AMP and 3'3'3'-cAAG at a distinct site from CDNs. One Acb2 hexamer can simultaneously bind two CTNs and three CDNs. Phage-encoded Acb2 provides protection from type III-C CBASS that uses cA3 signaling molecules. Moreover, phylogenetic analysis of >2,000 Acb2 homologs encoded by diverse phages and prophages revealed that most are expected to bind both CTNs and CDNs. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

Sensing of Bacterial Cyclic Dinucleotides by the Oxidoreductase RECON Promotes NF-κB Activation and Shapes a Proinflammatory Antibacterial State.

Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING signaling pathway, although evidence suggests that alternative pathways exist. We used cdN-conjugated beads to biochemically isolate host receptors for bacterial cdNs, and we identified the oxidoreductase RECON. High-affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and cosubstrate sites, as revealed by structural analyses. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. Bacterial infection of hepatocytes, which do not express STING, revealed that RECON negatively regulates NF-κB activation. Loss of RECON activity, via genetic ablation or inhibition by cdNs, increased NF-κB activation and reduced bacterial survival, suggesting that cdN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the antiviral state associated with STING activation. Thus, RECON functions as a cytosolic sensor for bacterial cdNs, shaping inflammatory gene activation via its effects on STING and NF-κB.

Cracking the DNA Code for V(D)J Recombination.

To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.

Structures of the P. aeruginosa FleQ-FleN master regulators reveal large-scale conformational switching in motility and biofilm control.

Pseudomonas aeruginosa can cause a wide array of chronic and acute infections associated with its ability to rapidly switch between planktonic, biofilm, and dispersed lifestyles, each with a specific arsenal for bacterial survival and virulence. At the cellular level, many of the physiological transitions are orchestrated by the intracellular second messenger c-di-GMP and its receptor-effector FleQ. A bacterial enhancer binding protein, FleQ acts as a master regulator of both flagellar motility and adherence factor secretion and uses remarkably different transcription activation mechanisms depending on its dinucleotide loading state, adenosine triphosphatase (ATPase) activity, interactions with polymerase sigma (σ) factors, and complexation with a second ATPase, FleN. How the FleQ-FleN tandem can exert diverse effects through recognition of a conserved FleQ binding consensus has remained enigmatic. Here, we provide cryogenic electron microscopy (cryo-EM) structures of both c-di-GMP-bound and c-di-GMP-free FleQ-FleN complexes which deepen our understanding of the proteins' (di)nucleotide-dependent conformational switching and fine-tuned roles in gene expression regulation.

The separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and the estimation of the binding constants and ionic mobilities of their complexes with 2-hydroxypropyl-ß-cyclodextrin by affinity capillary electrophoresis.

The incorporation of phosphorothioate linkages has recently been extensively employed in therapeutic oligonucleotides. For their separation and quality control, new high-efficient and high-sensitive analytical methods are needed. In this work, a new affinity capillary electrophoresis method has been developed and applied for the separation of a potential anticancer drug, 2',3'-cyclic diadenosine diphosphorothioate (Rp, Rp) (ADU-S100), and three recently newly synthesized diastereomers of its difluorinated derivative, 3',3'-cyclic di(2'-fluoro, 2'-deoxyadenosine phosphorothioate). The separation was performed in the various background electrolytes (BGEs) within a pH range 5-9 using several native and derivatized cyclodextrins (CDs) as chiral additives of the BGE. Relatively good separations were obtained with ß-, γ-, and 2-hydroxypropyl-γ-CDs in some of the BGEs tested. However, the best separation was achieved using the 2-hydroxypropyl-ß-CD chiral selector at 43.5 mM average concentration in the BGE composed of 40 mM Tris, 40 mM tricine, pH 8.1. Under these conditions, all the previous four cyclic dinucleotides (CDNs) were baseline separated within 4 min. Additionally, the average apparent binding constants and the average actual ionic mobilities of the complexes of all four CDNs with 2-hydroxypropyl-ß-CD in the above BGE were determined. The formed complexes were found to be relatively weak, with the average apparent binding constants in the range of 12.2-94.1 L mol-1 and with the actual ionic mobilities spanning the interval (-7.8 to -12.7) × 10-9 m2 V-1 s-1. The developed method can be applied for the separation, analysis, and characterization of the above and similar CDNs.

Chemo-enzymatic synthesis of NPN cofactor taking advantage of ADP-ribosyl cyclase and LarC cyclometallase promiscuous activities.

The nickel-pincer nucleotide cofactor (NPN) is a widespread organometallic cofactor required for lactate racemase (LarA) and for α-hydroxy acid racemases and epimerases of the LarA superfamily. Its biosynthesis, which starts with nicotinic acid adenine dinucleotide (NaAD), requires three enzymes: LarB, LarC, and LarE, and can be performed in vitro with purified enzymes. Nevertheless, as LarE and LarC are single turnover enzymes, the in vitro NPN biosynthesis requires huge amounts of enzymes (particularly 2 equivalents of LarE), which hampers the study of NPN and of NPN-dependent enzymes. By using adenosine diphosphate (ADP)-ribosyl cyclase (ARC), we exchanged the nicotinamide moiety in NAD+ with synthetic pyridine-3,5-bisthiocarboxylic acid in order to synthesize the novel intermediate pyridinium-3,5-bisthiocarboxylic acid adenine dinucleotide (P2TAD). The latter could be produced at a multimilligram scale allowing its characterization by Nuclear Magnetic Resonance (NMR) and mass spectrometry. Interestingly, P2TAD could directly be used by LarC in order to generate the NPN cofactor, bypassing both LarB and LarE. Globally, a new chemoenzymatic route towards NPN was developed via the intermediate P2TAD, which should facilitate the biochemical and biotechnological investigations on NPN binding enzymes.

Adaptation of a Commercial NAD+ Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1.

Here, we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3-acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base-exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base-exchange activity.

Evolution of cyclic di-GMP signalling on a short and long term time scale.

Diversifying radiation of domain families within specific lineages of life indicates the importance of their functionality for the organisms. The foundation for the diversifying radiation of the cyclic di-GMP signalling network that occurred within the bacterial kingdom is most likely based in the outmost adaptability, flexibility and plasticity of the system. Integrative sensing of multiple diverse extra- and intracellular signals is made possible by the N-terminal sensory domains of the modular cyclic di-GMP turnover proteins, mutations in the protein scaffolds and subsequent signal reception by diverse receptors, which eventually rewires opposite host-associated as well as environmental life styles including parallel regulated target outputs. Natural, laboratory and microcosm derived microbial variants often with an altered multicellular biofilm behaviour as reading output demonstrated single amino acid substitutions to substantially alter catalytic activity including substrate specificity. Truncations and domain swapping of cyclic di-GMP signalling genes and horizontal gene transfer suggest rewiring of the network. Presence of cyclic di-GMP signalling genes on horizontally transferable elements in particular observed in extreme acidophilic bacteria indicates that cyclic di-GMP signalling and biofilm components are under selective pressure in these types of environments. On a short and long term evolutionary scale, within a species and in families within bacterial orders, respectively, the cyclic di-GMP signalling network can also rapidly disappear. To investigate variability of the cyclic di-GMP signalling system on various levels will give clues about evolutionary forces and discover novel physiological and metabolic pathways affected by this intriguing second messenger signalling system.

Acidity constants and protonation sites of cyclic dinucleotides determined by capillary electrophoresis, quantum chemical calculations, and NMR spectroscopy.

Cyclic dinucleotides (CDNs) are important second messengers in bacteria and eukaryotes. Detailed characterization of their physicochemical properties is a prerequisite for understanding their biological functions. Herein, we examine acid-base and electromigration properties of selected CDNs employing capillary electrophoresis (CE), density functional theory (DFT), and nuclear magnetic resonance (NMR) spectroscopy to provide benchmark pKa values, as well as to unambiguously determine the protonation sites. Acidity constants (pKa ) of the NH+ moieties of adenine and guanine bases and actual and limiting ionic mobilities of CDNs were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities measured by CE in aqueous background electrolytes in a wide pH range (0.98-11.48), at constant temperature (25°C), and constant ionic strength (25 mM). The thermodynamic pKa values were found to be in the range 3.31-4.56 for adenine and 2.28-3.61 for guanine bases, whereas the pKa of enol group of guanine base was in the range 10.21-10.40. Except for systematic shifts of ∼2 pKa , the pKa values calculated by the DFT-D3//COSMO-RS composite protocol that included large-scale conformational sampling and "cross-morphing" were in a relatively good agreement with the pKa s determined by CE and predict N1 atom of adenine and N7 atom of guanine as the protonation sites. The protonation of the N1 atom of adenine and N7 atom of guanine in acidic background electrolytes (BGEs) and the dissociation of the enol group of guanine in alkaline BGEs was confirmed also by NMR spectroscopy.

Negative CG dinucleotide bias: An explanation based on feedback loops between Arginine codon assignments and theoretical minimal RNA rings.

Theoretical minimal RNA rings are candidate primordial genes evolved for non-redundant coding of the genetic code's 22 coding signals (one codon per biogenic amino acid, a start and a stop codon) over the shortest possible length: 29520 22-nucleotide-long RNA rings solve this min-max constraint. Numerous RNA ring properties are reminiscent of natural genes. Here we present analyses showing that all RNA rings lack dinucleotide CG (a mutable, chemically instable dinucleotide coding for Arginine), bearing a resemblance to known CG-depleted genomes. CG in "incomplete" RNA rings (not coding for all coding signals, with only 3-12 nucleotides) gradually decreases towards CG absence in complete, 22-nucleotide-long RNA rings. Presumably, feedback loops during RNA ring growth during evolution (when amino acid assignment fixed the genetic code) assigned Arg to codons lacking CG (AGR) to avoid CG. Hence, as a chemical property of base pairs, CG mutability restructured the genetic code, thereby establishing itself as genetically encoded biological information.

Novel nanoadjuvants balance immune activation with modest inflammation: implications for older adult vaccines.

BACKGROUND: Age-associated impairments of immune response and inflammaging likely contribute to poor vaccine efficacy. An appropriate balance between activation of immune memory and inflammatory response may be more effective in vaccines for older adults; attempts to overcome reduced efficacy have included the addition of adjuvants or increased antigenic dose. Next generation vaccine formulations may also use biomaterials to both deliver and adjuvant vaccine antigens. In the context of aging, it is important to determine the degree to which new biomaterials may enhance antigen-presenting cell (APC) functions without inducing potent inflammatory responses of APCs or other immune cell types (e.g., T cells). However, the effect of newer biomaterials on these cell types from young and older adults remains unknown. RESULTS: In this pilot study, cells from young and older adults were used to evaluate the effect of novel biomaterials such as polyanhydride nanoparticles (NP) and pentablock copolymer micelles (Mi) and cyclic dinucleotides (CDN; a STING agonist) on cytokine and chemokine secretion in comparison to standard immune activators such as lipopolysaccharide (LPS) and PMA/ionomycin. The NP treatment showed adjuvant-like activity with induction of inflammatory cytokines, growth factors, and select chemokines in peripheral blood mononuclear cells (PBMCs) of both young (n = 6) and older adults (n = 4), yet the degree of activation was generally less than LPS. Treatment with Mi or CDN resulted in minimal induction of cytokines and chemokine secretion with the exception of increased IFN-α and IL-12p70 by CDN. Age-related decreases were observed across multiple cytokines and chemokines, yet IFN-α, IL-12, and IL-7 production by NP or CDN stimulation was equal to or greater than in cells from younger adults. Consistent with these results in aged humans, a combination nanovaccine composed of NP, Mi, and CDN administered to aged mice resulted in a greater percentage of antigen-specific CD4+ T cells and greater effector memory cells in draining lymph nodes compared to an imiquimod-adjuvanted vaccine. CONCLUSIONS: Overall, our novel biomaterials demonstrated a modest induction of cytokine secretion with a minimal inflammatory profile. These findings suggest a unique role for biomaterial nanoadjuvants in the development of next generation vaccines for older adults.

Cytosolic detection of phagosomal bacteria-Mechanisms underlying PAMP exodus from the phagosome into the cytosol.

The metazoan innate immune system senses bacterial infections by detecting highly conserved bacterial molecules, termed pathogen-associated molecular patterns (PAMPs). PAMPs are detected by a variety of host pattern recognition receptors (PRRs), whose function is to coordinate downstream immune responses. PRR activities are, in part, regulated by their subcellular localizations. Accordingly, professional phagocytes can detect extracellular bacteria and their PAMPs via plasma membrane-oriented PRRs. Conversely, phagocytosed bacteria and their PAMPs are detected by transmembrane PRRs oriented toward the phagosomal lumen. Even though PAMPs are unable to passively diffuse across membranes, phagocytosed bacteria are also detected by PRRs localized within the host cell cytosol. This phenomenon is explained by phagocytosis of bacteria that specialize in phagosomal escape and cytosolic residence. Contrary to this cytosolic lifestyle, most bacteria studied to date spend their entire intracellular lifestyle contained within phagosomes, yet they also stimulate cytosolic PRRs. Herein, we will review our current understanding of how phagosomal PAMPs become accessible to cytosolic PRRs, as well as highlight knowledge gaps that should inspire future investigations.

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide.

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.

Synthesis of Mixed Dinucleotides by Mechanochemistry.

We report the synthesis of vitamin B1, B2, and B3 derived nucleotides and dinucleotides generated either through mechanochemical or solution phase chemistry. Under the explored conditions, adenosine and thiamine proved to be particularly amenable to milling conditions. Following optimization of the chemistry related to the formation pyrophosphate bonds, mixed dinucleotides of adenine and thiamine (vitamin B1), riboflavin (vitamin B2), nicotinamide riboside and 3-carboxamide 4-pyridone riboside (both vitamin B3 derivatives) were generated in good yields. Furthermore, we report an efficient synthesis of the MW+4 isotopologue of NAD+ for which deuterium incorporation is present on either side of the dinucleotidic linkage, poised for isotopic tracing experiments by mass spectrometry. Many of these mixed species are novel and present unexplored possibilities to simultaneously enhance or modulate cofactor transporters and enzymes of independent biosynthetic pathways.

DNA Modifications: Naturally More Error Prone?

Epigenetic DNA modifications are essential for normal cell function in vertebrates, but they can also be hotspots of mutagenesis. Methylcytosine in particular has long been known to be less stable than other nucleotides and spontaneously deaminates to thymine. Beyond this well-established phenomenon, however, the influence of epigenetic marks on mutagenesis has recently become an active field of investigation. In this review, we summarize current knowledge of the interactions between different DNA modifications and other mutagenic processes. External mutagens, such as UV light or smoking carcinogens, affect modified cytosines differently from unmodified ones, and modified cytosine can in some cases be protective rather than mutagenic. Notably, cell-intrinsic processes, such as DNA replication, also appear to influence the mutagenesis of modified cytosines. Altogether, evidence is accumulating to show that epigenetic changes have a profound influence on tissue-specific mutation accumulation.

The analog of cGAMP, c-di-AMP, activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells.

Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negative breast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1 cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.

A Luminescence-Based Coupled Enzyme Assay Enables High-Throughput Quantification of the Bacterial Second Messenger 3'3'-Cyclic-Di-AMP.

Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.

Cyclic di-AMP: Small molecule with big roles in bacteria.

Cyclic dinucleotides are second messengers that are present in all the three domains of life, bacteria, archaea, and eukaryotes. These dinucleotides have important physiological and pathophysiological roles in bacteria. Cyclic di-AMP (cdA) is one of the recently discovered cyclic dinucleotides present predominantly in gram-positive bacteria. cdA is synthesized through diadenylate cyclase (DAC) activity from ATP in a two-step process and hydrolyzed to linear dinucleotide pApA (and to 5' AMP in certain cases) by specific phosphodiesterases. cdA regulates various physiological processes like K+ transport and osmotic balance, DNA repair, cell wall homeostasis, drug resistance, central metabolism either by binding directly to the target protein or regulating its expression. It also participates in host-pathogen interaction by binding to host immune receptors ERAdP, RECON, and STING.

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity.

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.