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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 -
Acta Tropica Aug 2022Dengue virus is a life-threatening virus and cases of dengue infection have been increasing steadily in the past decades causing millions of deaths every year. So far,...
PURPOSE
Dengue virus is a life-threatening virus and cases of dengue infection have been increasing steadily in the past decades causing millions of deaths every year. So far, there is no vaccine that works effectively on all serotypes. Recently, CpG-recoded vaccines have proved to be effective against few viruses.
METHODS
In this study, evaluation and interpretation of more than 4547 Dengue virus genome sequences were included for analyzing novel CpG dinucleotides rich regions which are shared amid all serotypes. Genomic regions of DENV were synonymously CpG recoded using in silico methods and analyzed for adaptation in both human and Aedes spp. hosts based on CAI scores.
RESULTS
The analysis mirrored that serotypes 1, 3, and 4 shared CpG islands present in common regions. DENV-2 CpG islands showed no similarity with any of the CpG islands present in other serotypes. While DENV-3 sequences were found to possess the maximum number of conserved CpG islands stretches; DENV-2 was found to possess the lowest number. We found that all serotypes (with an exception of serotype 2) have CpG island in their 3' UTR. In silico CpG recoding of DENV genomic regions resulted in ∼ 3 fold increase of CpG dinucleotide frequency and comparative analysis based on CAI scores showed decreased adaptive fitness of CpG recoded DENV inside human host.
CONCLUSION
These CG-dinucleotide-enriched RNA sequences can be targeted by ZAP (zinc-finger antiviral protein) which can differentiate between host mRNA and viral mRNA. Our in silico findings can further be exploited for CpG-recoding of DENV genomes which can evoke cellular and humoral immune responses by recruiting ZAP-induced RNA degradation machinery and hence providing a promising approach for vaccine development.
Topics: Dengue; Dengue Virus; Dinucleoside Phosphates; Genomics; Humans; RNA, Messenger
PubMed: 35513073
DOI: 10.1016/j.actatropica.2022.106501 -
Arteriosclerosis, Thrombosis, and... Mar 2016
Topics: Animals; Blood Platelets; Coronary Thrombosis; Dinucleoside Phosphates; Female; Fibrinolytic Agents; Humans; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12
PubMed: 26912743
DOI: 10.1161/ATVBAHA.115.307097 -
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 -
International Journal of Molecular... Dec 2022Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated...
Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.
Topics: Nucleosides; Phosphates; Hydrolysis; Diphosphates; Chalcogens
PubMed: 36555224
DOI: 10.3390/ijms232415582 -
Scientific Reports Jul 2022In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium...
In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Dinucleoside Phosphates; Drug Tolerance; Mycobacterium smegmatis; Phenotype
PubMed: 35907936
DOI: 10.1038/s41598-022-17051-z -
Chemistry (Weinheim An Der Bergstrasse,... May 2020Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are...
Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen-associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides. The highly favorable photophysical features of the CDN analogues, when compared to their non-emissive natural counterparts, are used to monitor in real time the dinucleotide cyclase-mediated synthesis and phosphodiesterase (PDE)-mediated hydrolysis of homodimeric and mixed CDNs, providing effective means to probe the activities of two classes of bacterial enzymes and insight into their biomolecular recognition and catalytic features.
Topics: Bacterial Proteins; Dinucleoside Phosphates; Immunity, Innate; Protein Biosynthesis; Vibrio cholerae
PubMed: 32157755
DOI: 10.1002/chem.202001194 -
Journal of Bacteriology Apr 2020Diadenosine tetraphosphate (ApA) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various...
Diadenosine tetraphosphate (ApA) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. ApA is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving ApA hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect levels of ApA in YqeK shows a catalytic efficiency toward ApA similar to that of the symmetrically cleaving ApA hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that and genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals. Elevation of ApA level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major ApA hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient ApA hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens , , and By identifying the player contributing to ApA homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.
Topics: Acid Anhydride Hydrolases; Adenosine Diphosphate; Amino Acid Sequence; Bacteria; Bacterial Proteins; Catalysis; Cloning, Molecular; Dinucleoside Phosphates; Kinetics; Multigene Family; Phylogeny; Sequence Alignment; Staphylococcus aureus
PubMed: 32152217
DOI: 10.1128/JB.00053-20