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RNA (New York, N.Y.) Feb 2023mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such...
mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such therapeutics are an attractive alternative for DNA-based vaccines since they provide material that can be used with no risk of genomic integration. Additionally, mRNA can be quite easily engineered to introduce modifications for different applications or to modulate its properties, for example, to increase translational efficiency or stability, which is not available for DNA vectors. Here, we describe the use of N2 modified dinucleotide cap analogs as components of mRNA transcripts. The compounds obtained showed very promising biological properties while incorporated into mRNA. The presented N2-guanine modifications within the cap structure ensure proper attachment of the dinucleotide to the transcripts in the IVT reaction, guarantees their incorporation only in the correct orientation, and enables highly efficient translation of mRNA both in the in vitro translation system and in human HEK293 cells.
Topics: Humans; RNA, Messenger; Protein Biosynthesis; RNA Cap Analogs; HEK293 Cells; Dinucleoside Phosphates; Vaccines
PubMed: 36418172
DOI: 10.1261/rna.079460.122 -
Frontiers in Immunology 2022Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through...
Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through recombination or genetic attenuation and/or boost with different vaccines. Recent years, it was revealed that BCG could elicit non-specific heterogeneous protection against other pathogens such as viruses through a process termed trained immunity. Previously, we constructed a recombinant BCG (rBCG-DisA) with elevated c-di-AMP as endogenous adjuvant by overexpressing di-adenylate cyclase of DisA, and found that rBCG-DisA induced enhanced immune responses by subcutaneous route in mice after infection. In this study, splenocytes from rBCG-DisA immunized mice by intravenous route (i.v) elicited greater proinflammatory cytokine responses to homologous and heterologous re-stimulations than BCG. After infection, rBCG-DisA immunized mice showed hallmark responses of trained immunity including potent proinflammatory cytokine responses, enhanced epigenetic changes, altered lncRNA expressions and metabolic rewiring in bone marrow cells and other tissues. Moreover, rBCG-DisA immunization induced higher levels of antibodies and T cells responses in the lung and spleen of mice after infection. It was found that rBCG-DisA resided longer than BCG in the lung of infected mice implying prolonged duration of vaccine efficacy. Then, we found that rBCG-DisA boosting could prolong survival of BCG-primed mice over 90 weeks against infection. Our findings provided experimental evidence that rBCG-DisA with c-di-AMP as endogenous adjuvant induced enhanced trained immunity and adaptive immunity. What's more, rBCG-DisA showed promising potential in prime-boost strategy against infection in adults.
Topics: Acyltransferases; Adenosine Monophosphate; Adjuvants, Immunologic; Adjuvants, Pharmaceutic; Animals; Antigens, Bacterial; BCG Vaccine; Cyclic AMP; Cytokines; Dinucleoside Phosphates; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Mycobacterium tuberculosis; Tuberculosis, Lymph Node; Vaccines, Synthetic
PubMed: 36081510
DOI: 10.3389/fimmu.2022.943667 -
Science Signaling Sep 2022To colonize the host and cause disease, the human enteropathogen must sense, respond, and adapt to the harsh environment of the gastrointestinal tract. We showed that...
To colonize the host and cause disease, the human enteropathogen must sense, respond, and adapt to the harsh environment of the gastrointestinal tract. We showed that the production and degradation of cyclic diadenosine monophosphate (c-di-AMP) were necessary during different phases of growth, environmental adaptation, and infection. The production of this nucleotide second messenger was essential for growth because it controlled the uptake of potassium and also contributed to biofilm formation and cell wall homeostasis, whereas its degradation was required for osmotolerance and resistance to detergents and bile salts. The c-di-AMP binding transcription factor BusR repressed the expression of genes encoding the compatible solute transporter BusAA-AB. Compared with the parental strain, a mutant lacking BusR was more resistant to hyperosmotic and bile salt stresses, whereas a mutant lacking BusAA was more susceptible. A short exposure of cells to bile salts decreased intracellular c-di-AMP concentrations, suggesting that changes in membrane properties induce alterations in the intracellular c-di-AMP concentration. A strain that could not degrade c-di-AMP failed to persist in a mouse gut colonization model as long as the wild-type strain did. Thus, the production and degradation of c-di-AMP in have pleiotropic effects, including the control of osmolyte uptake to confer osmotolerance and bile salt resistance, and its degradation is important for host colonization.
Topics: Animals; Bacterial Proteins; Bile Acids and Salts; Clostridioides; Clostridioides difficile; Dinucleoside Phosphates; Humans; Mice
PubMed: 36067333
DOI: 10.1126/scisignal.abn8171 -
Open Heart Aug 2022To estimate the incidence and HRs for bleeding for different dual antiplatelet therapies (DAPT) in a real-world population with acute coronary syndrome (ACS) undergoing... (Randomized Controlled Trial)
Randomized Controlled Trial
Real-world bleeding in patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) and prescribed different combinations of dual antiplatelet therapy (DAPT) in England: a population-based cohort study emulating a 'target trial'.
OBJECTIVE
To estimate the incidence and HRs for bleeding for different dual antiplatelet therapies (DAPT) in a real-world population with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) in England.
DESIGN
A retrospective, population-based cohort study emulating a target randomised controlled trial (tRCT).
DATA SOURCES
Linked Clinical Practice Research Datalink (CPRD) and Hospital Episode Statistics (HES).
SETTING
Primary and secondary care.
PARTICIPANTS
Patients ≥18 years old with ACS undergoing emergency PCI.
INTERVENTIONS
Aspirin and clopidogrel (AC, reference) versus aspirin and prasugrel (AP) or aspirin and ticagrelor (AT); AP evaluated only in patients with ST-elevation myocardial infarction (STEMI).
MAIN OUTCOME MEASURES
Primary: any bleeding up to 12 months after the index event (HES- or CPRD- recorded). Secondary: HES-recorded bleeding, CPRD-recorded bleeding, all-cause and cardiovascular mortality, mortality from bleeding, myocardial infarction, stroke, additional coronary intervention and major adverse cardiovascular and cerebrovascular events (MACCE).
RESULTS
In ACS, the rates of any bleeding for AC and AT were 89 per 1000 person years and 134 per 1000 person years, respectively. In STEMI, rates for AC, AP and AT were 93 per 1000 person years, 138 per 1000 person years and 143 per 100 person years, respectively. In ACS, compared with AC, AT increased the hazard of any bleeding (HR: 1.47, 95% CI 1.19 to 1.82) but did not reduce MACCE (HR: 1.06, 95% CI 0.89 to 1.27). In STEMI, compared with AC, AP and AT increased the hazard of any bleeding (HR: 1.77, 95% CI 1.21 to 2.59 and HR: 1.50, 95% CI 1.10 to 2.05, respectively) but did not reduce MACCE (HR: 1.10, 95% CI 0.80 to 1.51 and HR: 1.21, 95% CI 0.94 to 1.51, respectively). Non-adherence to the prescribed DAPT regimen was 28% in AC (29% in STEMI only), 31% in AP (STEMI only) and 33% in AT (32% in STEMI only).
CONCLUSIONS
In a real-world population with ACS, DAPT with ticagrelor or prasugrel are associated with increased bleeding compared with DAPT with clopidogrel.
TRIAL REGISTRATION NUMBER
ISRCTN76607611.
Topics: Acute Coronary Syndrome; Adolescent; Aspirin; Clopidogrel; Cohort Studies; Dinucleoside Phosphates; Hemorrhage; Humans; Percutaneous Coronary Intervention; Platelet Aggregation Inhibitors; Prasugrel Hydrochloride; Retrospective Studies; ST Elevation Myocardial Infarction; Ticagrelor
PubMed: 35961692
DOI: 10.1136/openhrt-2022-001999 -
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 -
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 -
Nature Aug 2022Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes....
Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide, 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 mammals. 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.
Topics: Animals; Antiviral Agents; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; Dinucleoside Phosphates; Humans; Immunity, Innate; Membrane Proteins; Operon; Receptors, Interleukin-1; Sphingobacterium; Toll-Like Receptors
PubMed: 35859168
DOI: 10.1038/s41586-022-04999-1 -
NPJ Biofilms and Microbiomes Jul 2022Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis....
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 (pde) 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 pde 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.
Topics: Adenosine Monophosphate; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Homeostasis; Phosphoric Diester Hydrolases; Porphyromonas gingivalis; Virulence
PubMed: 35794154
DOI: 10.1038/s41522-022-00316-w -
Communications Biology Jul 2022Predicting protein-protein interaction and non-interaction are two important different aspects of multi-body structure predictions, which provide vital information about...
Predicting protein-protein interaction and non-interaction are two important different aspects of multi-body structure predictions, which provide vital information about protein function. Some computational methods have recently been developed to complement experimental methods, but still cannot effectively detect real non-interacting protein pairs. We proposed a gene sequence-based method, named NVDT (Natural Vector combine with Dinucleotide and Triplet nucleotide), for the prediction of interaction and non-interaction. For protein-protein non-interactions (PPNIs), the proposed method obtained accuracies of 86.23% for Homo sapiens and 85.34% for Mus musculus, and it performed well on three types of non-interaction networks. For protein-protein interactions (PPIs), we obtained accuracies of 99.20, 94.94, 98.56, 95.41, and 94.83% for Saccharomyces cerevisiae, Drosophila melanogaster, Helicobacter pylori, Homo sapiens, and Mus musculus, respectively. Furthermore, NVDT outperformed established sequence-based methods and demonstrated high prediction results for cross-species interactions. NVDT is expected to be an effective approach for predicting PPIs and PPNIs.
Topics: Animals; Dinucleoside Phosphates; Drosophila melanogaster; Genetic Techniques; Genetic Vectors; Helicobacter pylori; Mice; Saccharomyces cerevisiae
PubMed: 35780196
DOI: 10.1038/s42003-022-03617-0 -
The Journal of Biological Chemistry Jul 2022The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and...
The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.
Topics: Adenine; Adenosine Monophosphate; Bacillus subtilis; Bacterial Proteins; Dinucleoside Phosphates
PubMed: 35714772
DOI: 10.1016/j.jbc.2022.102144