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Nature Communications Feb 2020It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpNs) and yet their roles and mechanisms of action remain unclear. Here, we show that...
It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpNs serve as RNA caps in Escherichia coli. NpNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpN-caps from RNA. ApaH is able to cleave all NpN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.
Topics: Acid Anhydride Hydrolases; DNA-Directed RNA Polymerases; Dinucleoside Phosphates; Escherichia coli; Escherichia coli Proteins; Methylation; Nucleic Acid Conformation; RNA Caps; RNA Stability; RNA, Bacterial
PubMed: 32103016
DOI: 10.1038/s41467-020-14896-8 -
Nature Communications Jan 2022Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense...
Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in β-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.
Topics: Bacteria; Crystallography, X-Ray; Cyclic GMP; Dinucleoside Phosphates; Humans; Immunity, Innate; Interferons; Ligands; Membrane Proteins; Nucleotidyltransferases; Prevotella
PubMed: 35013136
DOI: 10.1038/s41467-021-26583-3 -
Chembiochem : a European Journal of... Apr 2022The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the...
The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-c GAMP and 3'3'-c GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-c GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-c GAMP in THP-1 cells.
Topics: Animals; DNA; Dinucleoside Phosphates; Humans; Interferons; Mammals; Membrane Proteins; Mice; Nucleotidyltransferases; Second Messenger Systems
PubMed: 35189023
DOI: 10.1002/cbic.202200005 -
Journal of Bacteriology Apr 2023Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to... (Review)
Review
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 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.
Topics: Humans; Archaea; Phosphorus-Oxygen Lyases; Bacterial Proteins; Bacteria; Second Messenger Systems; Cyclic AMP; Dinucleoside Phosphates
PubMed: 37022175
DOI: 10.1128/jb.00023-23 -
Current Diabetes Reports Jun 2023Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. The majority of diabetes deaths (> 80%) occur in low- and... (Review)
Review
PURPOSE OF REVIEW
Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. The majority of diabetes deaths (> 80%) occur in low- and middle-income countries, which are predominant in Latin America. Therefore, the purpose of this article is to compare the clinical practice guideline (CPG) for the pharmacological management of T2DM in Latin America (LA) with international reference guidelines.
RECENT FINDINGS
Several LA countries have recently developed CPGs. However, the quality of these guidelines is unknown according to the AGREE II tool and taking as reference three CPGs of international impact: American Diabetes Association (ADA), European Diabetes Association (EASD), and Latin American Diabetes Association (ALAD). Ten CPGs were selected for analysis. The ADA scored > 80% on the AGREE II domains and was selected as the main comparator. Eighty percent of LA CPGs were developed before 2018. Only one was not recommended (all domains < 60%). The CPGs in LA have good quality but are outdated. They have significant gaps compared to the reference. There is a need for improvement, as proposing updates every three years to maintain the best available clinical evidence in all guidelines.
Topics: Humans; Diabetes Mellitus, Type 2; Latin America; Dinucleoside Phosphates
PubMed: 37126189
DOI: 10.1007/s11892-023-01504-4 -
Biophysical Journal Jun 2022Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts...
Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant K for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.
Topics: Bacterial Proteins; Dinucleoside Phosphates; Flavins; Flavoproteins; Gene Expression Regulation, Bacterial; Heme; Repressor Proteins; Rhodobacter sphaeroides
PubMed: 35488435
DOI: 10.1016/j.bpj.2022.04.031 -
Nature Microbiology Sep 2022Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the...
Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.
Topics: Bacillus subtilis; Dinucleoside Phosphates; Guanosine Triphosphate
PubMed: 35953658
DOI: 10.1038/s41564-022-01193-x -
Analytical and Bioanalytical Chemistry Nov 2021Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from...
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
PubMed: 34476522
DOI: 10.1007/s00216-021-03628-6 -
Frontiers in Immunology 2019Cyclic-di-AMP (c-di-AMP) is a bacterial second messenger that is produced by intracellular bacterial pathogens in mammalian host macrophages. Previous reports have shown...
Cyclic-di-AMP (c-di-AMP) is a bacterial second messenger that is produced by intracellular bacterial pathogens in mammalian host macrophages. Previous reports have shown that c-di-AMP is recognized by intracellular pattern recognition receptors of the innate immune system and stimulate type I interferon response. Here we report that the response to c-di-AMP includes a post-transcriptional component that is involved in the induction of additional inflammatory cytokines including IL-6, CXCL2, CCL3, and CCL4. Their mRNAs contain AU-rich elements (AREs) in their 3' UTR that promote decay and repress translation. We show that c-di-AMP leads to the phosphorylation of p38 MAPK as well as the induction of the ARE-binding protein TTP, both of which are components of a signaling pathway that modulate the expression of ARE-containing mRNAs at the post-transcriptional level. Pharmacological inhibition of p38 reduces the c-di-AMP-dependent release of induced cytokines, while TTP knockdown increases their release and mRNA stability. C-di-AMP can specifically increase the expression of a nano-Luciferase reporter that contains AREs. We propose a non-canonical intracellular mode of activation of the p38 MAPK pathway with the subsequent enhancement in the expression of inflammatory cytokines. C-di-AMP is widely distributed in bacteria, including infectious intracellular pathogens; hence, understanding of its post-transcriptional gene regulatory effect on the host response may provide novel approaches for therapy.
Topics: 3' Untranslated Regions; AU Rich Elements; Animals; Bacteria; Bacterial Infections; Cytokines; Dinucleoside Phosphates; Gene Expression Regulation; Genes, Reporter; Host-Pathogen Interactions; Mice; Open Reading Frames; Promoter Regions, Genetic; RAW 264.7 Cells; RNA Processing, Post-Transcriptional; RNA Stability; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 32010134
DOI: 10.3389/fimmu.2019.03050 -
Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection.Cell Host & Microbe Jul 2022Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes...
Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.
Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Folic Acid Antagonists; Inflammation; Mice; Staphylococcal Infections; Staphylococcus aureus; Thymidine
PubMed: 35439435
DOI: 10.1016/j.chom.2022.03.028