Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates ...stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program.These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.
Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a ...specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
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•REXO2 is a specialized dinucleotidase present in mammalian mitochondria•REXO2 is essential for embryonic development in mice•Dinucleotides stimulate mitochondrial transcription in vitro and in vivo•Dinucleotide degradation is required to prevent their use as transcription primers
Nicholls et al. study the function of the human oligoribonuclease REXO2 and find that it is a specialized dinucleotidase. The Rexo2 gene is essential for embryonic development, and its conditional loss results in changes to mitochondrial transcription patterns, indicating the use of dinucleotides for promoter-independent mitochondrial transcription initiation.
Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the ...functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2′,3′- and 3′,5′-cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the σD-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.
Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial ...cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY(+)) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular (PMVECs) endothelial cells both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP ≈ cGMP ≈ cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY(+) intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h, when the experiment was terminated. cGMP concentrations increased within 1 h postinfection, whereas cAMP and cUMP concentrations increased within 3 h, and cCMP concentrations increased within 4 h postinfection. In PMVECs, intercellular gaps did not form until 4 h postinfection. Only cGMP and cUMP concentrations increased at 3 and 6 h postinfection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY(+) intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.
AIMS: To develop new genetic tools for studying 3′,5′‐cyclic diguanylic acid (c‐di‐GMP) signalling in Pseudomonas aeruginosa. METHODS AND RESULTS: Plasmid pPcdrA::lux, carrying a transcriptional ...fusion between the c‐di‐GMP responsive promoter PcdrA and the luxCDABE reporter genes, has been generated and validated in purpose‐built P. aeruginosa strains in which c‐di‐GMP levels can be increased or reduced upon arabinose‐dependent induction of c‐di‐GMP synthetizing or degrading enzymes. CONCLUSIONS: The reporter systems described so far were able to detect a decrease in the c‐di‐GMP levels only in engineered strains overproducing c‐di‐GMP. Conversely, pPcdrA::lux could be used for studying any process or chemical compound expected to cause both an increase or a decrease with respect to the c‐di‐GMP levels produced by wild type P. aeruginosa. Another relevant aspect of this study has been the development of novel and improved genetic devices for the fine arabinose‐dependent control of c‐di‐GMP levels in P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic tools developed and validated in this study could facilitate investigations tackling the c‐di‐GMP signalling process on different fields, from cellular physiology to drug‐discovery research.
Donor alloantigen infusion induces T cell regulation and transplant tolerance in small animals. Here, we study donor splenocyte infusion in a large animal model of pulmonary transplantation. Major ...histocompatibility complex–mismatched single lung transplantation was performed in 28 minipigs followed by a 28‐day course of methylprednisolone and tacrolimus. Some animals received a perioperative donor or third party splenocyte infusion, with or without low‐dose irradiation (IRR) before surgery. Graft survival was significantly prolonged in animals receiving both donor splenocytes and IRR compared with controls with either donor splenocytes or IRR only. In animals with donor splenocytes and IRR, increased donor cell chimerism and CD4+CD25high+ T cell frequencies were detected in peripheral blood associated with decreased interferon‐γ production of leukocytes. Secondary third‐party kidney transplants more than 2 years after pulmonary transplantation were acutely rejected despite maintained tolerance of the lung allografts. As a cellular control, additional animals received third‐party splenocytes or donor splenocyte protein extracts. While animals treated with third‐party splenocytes showed significant graft survival prolongation, the subcellular antigen infusion showed no such effect. In conclusion, minipigs conditioned with preoperative IRR and donor, or third‐party, splenocyte infusions may develop long‐term donor‐specific pulmonary allograft survival in the presence of high levels of circulating regulatory T cells.
Early leukocyte chimerism increases with donor spleen cell infusion following low‐dose recipient irradiation in an outbred miniature swine lung transplantation model, resulting in long‐term lung allograft survival but preserved immune competence versus delayed third‐party kidney allografts.
The immunosuppressant sirolimus and curcumin, the main principle of the turmeric spice, have shown antiproliferative effects on many human and not-human cell lines. Whereas the antiproliferative ...effect of sirolimus is mainly mediated by inhibition of mTOR, curcumin is described to affect many molecular targets which makes it unpredictable to appraise if the effects of these both substances on cell proliferation and especially on immunosuppression are additive or synergistic. To answer this question we investigated the interaction of both these substances on OKT3-induced human peripheral blood mononuclear cell (PBMC) proliferation. OKT3-induced human PBMC proliferation was determined by measuring 3H-thymidine incorporation. Influence of curcumin on interleukin-2 (IL-2) release and IκB-phosphorylation in PBMC was determined by ELISA and western blot, respectively. Curcumin-induced apoptosis and necrosis was analyzed by FACS analysis. Whereas curcumin completely inhibited OKT3-induced PBMC proliferation in a dose-dependent manner with an IC50 of 2.8μM, sirolimus could reduce PBMC proliferation dose-dependently only to a minimum of 28% at a concentration of 5ng/ml (IC50 1.1ng/ml). When curcumin was combined at concentrations of 1.25–2.5μM with sirolimus at concentrations from 0.63 to 1.25ng/ml the effects were synergistic. Combination of curcumin (1.25–2.5μM) with sirolimus (5ng/ml) showed additive effects. The effects after combination of curcumin at 5μM with each sirolimus concentration and sirolimus at 10ng/ml with each curcumin concentration were presumably antagonistic. We conclude that the immunosuppressive effects of curcumin and sirolimus in low concentrations are synergistic in OKT3-activated PBMC. Whether curcumin and sirolimus have also synergistic antiproliferative effects in tumor cells has to be shown in further experiments including animal models.
Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as ...biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in
O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain
ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (
ed,
ry,
nd
ough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of
mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene
Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3'3'-cGAMP.
, only the 3'3'-cGAMP concentration was elevated upon short-term overexpression of
, making this work a first report on cGAMP production in
Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in
ECOR31.
The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the
biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal
strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.
The alkaloid sinomenine extracted from the medicinal plant Sinomenium acutum is used in China for the treatment of various rheumatic diseases. It has immunomodulatory properties in a cardiac ...allograft transplantation model. Its antiproliferative effect on human mononuclear cells in combination with different immunosuppressive drugs was further analysed in vitro. Sinomenine dose-dependently attenuated thymidine incorporation, interleukin-2 synthesis, and cell cycle progression of activated T-lymphocytes. Cell proliferation was synergistically decreased by addition of sinomenine together with suboptimal concentrations of the established immunosuppressive drugs tacrolimus or mycophenolic acid, respectively.
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