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Proceedings of the National Academy of... Mar 2020Antibiotic-producing use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and...
Antibiotic-producing use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as and AtaC is monomeric in solution and binds Mn to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in , connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in .
Topics: Adenosine Monophosphate; Bacterial Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Hydrolysis; Mycobacterium tuberculosis; Phosphoric Diester Hydrolases; Second Messenger Systems; Signal Transduction; Streptococcus pneumoniae; Streptomyces
PubMed: 32188788
DOI: 10.1073/pnas.1917080117 -
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 -
Nature Communications Oct 2021The nucleotides diadenosine triphosphate (ApA) and diadenosine tetraphosphate (ApA) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase...
The nucleotides diadenosine triphosphate (ApA) and diadenosine tetraphosphate (ApA) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of ApA and ApA and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApAs across the domains of life, but also identify those that are different for ApA or ApA. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.
Topics: Adenosine Triphosphate; Dinucleoside Phosphates; Endoribonucleases; Escherichia coli; Escherichia coli Proteins; HEK293 Cells; Humans; L-Lactate Dehydrogenase; Phosphoglycerate Kinase; Photoaffinity Labels; Protein Binding; Proteomics; Ubiquitin-Activating Enzymes
PubMed: 34608152
DOI: 10.1038/s41467-021-26075-4 -
BMC Evolutionary Biology Feb 2020Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It...
BACKGROUND
Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It also has the highest GC content of any chromosome, especially outside gene clusters. The high GC content and concomitant high content of hypermutable CpG sites raises the possibility chromosome 19 exhibits higher levels of nucleotide diversity both within and between species, and may possess greater variation in DNA methylation that regulates gene expression.
RESULTS
We examined GC and CpG content of chromosome 19 orthologs across representatives of the primate order. In all 12 primate species with suitable genome assemblies, chromosome 19 orthologs have the highest GC content of any chromosome. CpG dinucleotides and CpG islands are also more prevalent in chromosome 19 orthologs than other chromosomes. GC and CpG content are generally higher outside the gene clusters. Intra-species variation based on SNPs in human common dbSNP, rhesus, crab eating macaque, baboon and marmoset datasets is most prevalent on chromosome 19 and its orthologs. Inter-species comparisons based on phyloP conservation show accelerated nucleotide evolution for chromosome 19 promoter flanking and enhancer regions. These same regulatory regions show the highest CpG density of any chromosome suggesting they possess considerable methylome regulatory potential.
CONCLUSIONS
The pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur representing 74 million years of primate evolution. Much CpG variation exists both within and between primate species with a portion of this variation occurring in regulatory regions.
Topics: Animals; Base Composition; Base Sequence; Chromosomes; Chromosomes, Human, Pair 19; Conserved Sequence; CpG Islands; DNA Methylation; Dinucleoside Phosphates; Genome; Humans; Lemur; Mice; Multigene Family; Phylogeny; Primates; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid
PubMed: 32106815
DOI: 10.1186/s12862-020-1595-9 -
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 -
Environmental Microbiology Jul 2020The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of...
The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.
Topics: Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Homeostasis; Listeria monocytogenes; Osmotic Pressure; Phosphoglucomutase
PubMed: 32250026
DOI: 10.1111/1462-2920.15008 -
The c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.The FEBS Journal Jun 2023Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly...
Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.
Topics: Carrier Proteins; Streptococcus agalactiae; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Potassium
PubMed: 36629470
DOI: 10.1111/febs.16724 -
Journal of the American Chemical Society Nov 2019Cyclic dinucleotides have emerged as important secondary messengers and cell signaling molecules that regulate several cell responses. A guanine-deficit G-quadruplex...
Cyclic dinucleotides have emerged as important secondary messengers and cell signaling molecules that regulate several cell responses. A guanine-deficit G-quadruplex structure formation by a sequence containing (4 - 1) guanines, denoting the number of G-tetrad layers, was previously reported. Here, a (4 - 1) G-quadruplex structure is shown to be capable of binding guanine-containing dinucleotides in micromolar affinity. The guanine base of the dinucleotides interacts with a vacant G-triad, forming four additional Hoogsteen hydrogen bonds to complete a G-tetrad. Solution structures of two complexes, both comprised of a (4 - 1) G-quadruplex structure, one bound to a linear dinucleotide (d(AG)) and the other to a cyclic dinucleotide (cGAMP), are solved using NMR spectroscopy. The latter suggests sufficiently strong interaction between the guanine base of the dinucleotide and the vacant G-triad, which acts as an anchor point of binding. The binding interfaces from the two solution structures provide useful information for specific ligand design. The results also infer that other guanine-containing metabolites of a similar size have the capability of binding G-quadruplexes, potentially affecting the expression of the metabolites and functionality of the bound G-quadruplexes.
Topics: Dinucleoside Phosphates; G-Quadruplexes; Guanine; Hydrogen Bonding
PubMed: 31661272
DOI: 10.1021/jacs.9b05642 -
The Science of the Total Environment Aug 2021DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) is a γ-secretase inhibitor that indirectly blocks the activity of Notch pathway. It is a...
DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) is a γ-secretase inhibitor that indirectly blocks the activity of Notch pathway. It is a potential therapeutic target drug for many diseases, such as cancer, neurological, cardiovascular, and cerebrovascular diseases. However, the pharmacological action and specific mechanisms of DAPT are not clear. Planarians have strong regenerative capacity and can regenerate a new individual with a complete nervous system in one week. Thus, they are used as an ideal indicator of environmental toxicants and a novel model for studying neurodevelopmental toxicology. In this study, different concentrations and treatment times of DAPT are used to analyze the gene expression levels of major components in Notch pathway. The results show that the optimal concentration and exposure time of DAPT is 100 nM for 10 days in planarians and indicate that the inhibitory of DAPT treatment on Notch pathway is time- and concentration-dependent. The potential impact of DAPT is effectively analyzed by qPCR, WISH, and Immunofluorescence. The results indicate that DAPT exposure causes intact planarian wavy or swollen, and regenerative planarians asymmetric growth or muti-eye. Moreover, DAPT exposure increases cell proliferation and apoptosis, results in neurodevelopmental defects and dynamic changes of some marker genes. These results suggest that the balance of proliferation and apoptosis is disturbed, and then, affecting tissue homeostasis and differentiation. These findings demonstrate that DAPT has serious side effects in organisms and relies on Notch pathway to determine cell fate, it is cautious in the use of DAPT as a potential therapeutic approach for the disease in clinical trials.
Topics: Amyloid Precursor Protein Secretases; Animals; Dinucleoside Phosphates; Pharmaceutical Preparations; Planarians; Signal Transduction
PubMed: 33812110
DOI: 10.1016/j.scitotenv.2021.146735 -
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