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The New Phytologist Jun 2022The amount of inorganic carbon (C ) fluctuates in aquatic environments. Cyanobacteria evolved a C -concentrating mechanism (CCM) that is regulated at different levels....
The amount of inorganic carbon (C ) fluctuates in aquatic environments. Cyanobacteria evolved a C -concentrating mechanism (CCM) that is regulated at different levels. The regulator SbtB binds to the second messengers cAMP or c-di-AMP and is involved in acclimation to low C (LC) in Synechocystis sp. PCC 6803. Here, we investigated the role of SbtB and of associated second messengers at different C conditions. The transcriptome of wild-type (WT) Synechocystis and the ΔsbtB mutant were compared with Δcya1, a mutant defective in cAMP production, and ΔdacA, a mutant defective in generating c-di-AMP. A defined subset of LC-regulated genes in the WT was already changed in ΔsbtB under high C (HC) conditions. This response of ΔsbtB correlated with a diminished induction of many CCM-associated genes after LC shift in this mutant. The Δcya1 mutant showed less deviation from WT, whereas ΔdacA induced CCM-associated genes under HC. Metabolome analysis also revealed differences between the strains, whereby ΔsbtB showed slower accumulation of 2-phosphoglycolate and ΔdacA differences among amino acids compared to WT. Collectively, these results indicate that SbtB regulates a subset of LC acclimation genes while c-di-AMP and especially cAMP appear to have a lesser impact on gene expression under different C availabilities.
Topics: Bacterial Proteins; Carbon; Carbon Dioxide; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Photosynthesis; Second Messenger Systems; Synechocystis; Transcriptome
PubMed: 35285042
DOI: 10.1111/nph.18094 -
Vaccine Apr 2022The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The...
The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi-challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8IFN-γ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8IFN-γ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi.
Topics: Animals; Chagas Disease; Dinucleoside Phosphates; Glycoproteins; Immunization; Mice; Mice, Inbred BALB C; Neuraminidase; Protozoan Vaccines; Trypanosoma cruzi
PubMed: 35279330
DOI: 10.1016/j.vaccine.2022.02.071 -
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 -
Nature Communications Feb 2022In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed...
In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.
Topics: Animals; BCG Vaccine; CD8-Positive T-Lymphocytes; Cancer Vaccines; Cell Line, Tumor; Cytokines; Dinucleoside Phosphates; Humans; Immunity, Innate; Macrophages; Membrane Proteins; Mice; Myeloid Cells; Pathogen-Associated Molecular Pattern Molecules; Rats; Urinary Bladder Neoplasms; Urothelium; Vaccination
PubMed: 35169141
DOI: 10.1038/s41467-022-28509-z -
Scientific Reports Feb 2022The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA...
The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.
Topics: Animals; Base Sequence; Binding Sites; COVID-19; Dinucleoside Phosphates; Evolution, Molecular; Genome, Viral; Host-Pathogen Interactions; Humans; Nucleotide Motifs; Protein Binding; RNA, Viral; RNA-Binding Proteins; SARS-CoV-2; Virus Replication
PubMed: 35165300
DOI: 10.1038/s41598-022-06046-5 -
MBio Feb 2021In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of...
In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both and . This interaction stimulates the activity of the enzyme, as demonstrated by enzyme assays and metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively. If bacteria experience a starvation for potassium, by far the most abundant metal ion in every living cell, they have to activate high-affinity potassium transporters, switch off growth activities such as translation and transcription of many genes or replication, and redirect the metabolism in a way that the most essential functions of potassium can be taken over by metabolites. Importantly, potassium starvation triggers a need for glutamate-derived amino acids. In many bacteria, the responses to changing potassium availability are orchestrated by a nucleotide second messenger, cyclic di-AMP. c-di-AMP binds to factors involved directly in potassium homeostasis and to dedicated signal transduction proteins. Here, we demonstrate that in the Gram-positive model organism Bacillus subtilis, the c-di-AMP receptor protein DarB can bind to and, thus, activate pyruvate carboxylase, the enzyme responsible for replenishing the citric acid cycle. This interaction takes place under conditions of potassium starvation if DarB is present in the apo form and the cells are in need of glutamate. Thus, DarB links potassium availability to the control of central metabolism.
Topics: Cyclic AMP; Bacillus subtilis; Pyruvate Carboxylase; Bacterial Proteins; Second Messenger Systems; Dinucleoside Phosphates; Glutamic Acid; Potassium
PubMed: 35130724
DOI: 10.1128/mbio.03602-21 -
Nature Immunology Feb 2022The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides...
The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2'3'cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c mice have increased cell cycle progression, proliferation, survival, Ca influx and cytokine production-a phenotype associated with downmodulation of p53 signaling. Mechanistically, LRRC8C mediates the transport of 2'3'cGAMP in T cells, resulting in STING and p53 activation. Inhibition of STING recapitulates the phenotype of LRRC8C-deficient T cells, whereas overexpression of p53 inhibits their enhanced T cell function. Lrrc8c mice have exacerbated T cell-dependent immune responses, including immunity to influenza A virus infection and experimental autoimmune encephalomyelitis. Our results identify cGAMP uptake through LRRC8C and STING-p53 signaling as a new inhibitory signaling pathway in T cells and adaptive immunity.
Topics: Animals; Anions; Calcium; Dinucleoside Phosphates; Female; Ion Channels; Membrane Proteins; Mice; Mice, Inbred C57BL; Nucleotides, Cyclic; Signal Transduction; T-Lymphocytes; Tumor Suppressor Protein p53
PubMed: 35105987
DOI: 10.1038/s41590-021-01105-x -
Nature Immunology Feb 2022
Topics: Anions; Dinucleoside Phosphates; T-Lymphocytes
PubMed: 35105986
DOI: 10.1038/s41590-021-01118-6 -
Cardiovascular Revascularization... Aug 2022Guidelines recommend individualization of dual antiplatelet therapy (DAPT) duration. Whether to guide decisions based on bleeding risk, ischemic risk or a combination is...
BACKGROUND
Guidelines recommend individualization of dual antiplatelet therapy (DAPT) duration. Whether to guide decisions based on bleeding risk, ischemic risk or a combination is not known.
AIMS
To compare a bleeding prediction model, an ischemic prediction model, and the DAPT score in guiding DAPT duration.
METHODS
11,648 patients in the DAPT Study were categorized into higher and lower risk using a bleeding model, an ischemic model, and the DAPT score. Effect of 30 vs. 12 months of DAPT on bleeding events, ischemic events, and the combination (net-adverse clinical events [NACE]) was assessed.
RESULTS
Among patients stratified with the bleeding model, 30 vs. 12 months of DAPT resulted in similar ischemic and bleeding event rates. With the ischemic model, however, higher risk patients had a greater reduction in ischemic events with extended duration of DAPT (difference in risk differences [DRD]: -2.6%, 95% CI: -3.9 to -1.3%; p < 0.01), and a smaller increase in bleeding (DRD: -1.0%, 95% CI: -2.1-0.0%; p = 0.04). Similarly, high DAPT score patients had a greater reduction in ischemic events with extended DAPT duration (DRD: -2.4%, 95%: CI: -3.6 to -1.1%; p < 0.01) and a smaller increase in bleeding (DRD: -1.2%, 95%: CI: -2.2-0.0%; p = 0.02). Although NACE was similar for bleeding risk groups, NACE was significantly reduced with extended DAPT in the higher ischemic risk and high DAPT score groups.
CONCLUSIONS
In this low-bleeding risk population, stratifying patients based on predicted ischemic risk and the DAPT score best discerned benefit versus harm of extended DAPT duration on ischemic events, bleeding events, and NACE.
CONDENSED ABSTRACT
Duration of dual antiplatelet therapy (DAPT) should be guided by an individualized risk assessment. Bleeding risk tools have emerged to identify patients at high bleeding risk for whom truncated DAPT therapy may be safest. In a lower bleeding risk population, however, whether DAPT duration should be guided by bleeding risk, ischemic risk, or a combination is unknown. In this analysis, implementation of a score based on ischemic risk prediction and the DAPT score (a combination of ischemic and bleeding risk) best predicted ischemic events, bleeding events, and net-adverse clinical events (NACE).
Topics: Dinucleoside Phosphates; Drug Therapy, Combination; Dual Anti-Platelet Therapy; Hemorrhage; Humans; Ischemia; Percutaneous Coronary Intervention; Platelet Aggregation Inhibitors; Treatment Outcome
PubMed: 35045941
DOI: 10.1016/j.carrev.2022.01.006 -
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