Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.
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ABSTRACT:
SUBMITTER: Li Y
PROVIDER: S-EPMC7494572 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cyclic dinucleotides (CDNs) have been previously recognized as important secondary signaling molecules in bacteria and, more recently, in mammalian cells. In the former case, they represent secondary messengers affecting numerous responses of the prokaryotic cell, whereas in the latter, they act as agonists of the innate immune response. Remarkable new discoveries have linked these two patterns of utilization of CDNs as secondary messengers and have revealed unexpected influences they likely had on shaping human genetic variation. This Review summarizes these recent insights and provides a perspective on future unanswered questions in this exciting field.
| S-EPMC3931520 | biostudies-literature
Project description:The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3'5'-3'5' cyclic GMP-AMP (3'3' cGAMP) produced by Vibrio cholerae and metazoan second messenger 2'5'-3'5' Cyclic GMP-AMP (2'3' cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-? and NF-?B promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-?signaling, while it was defective for the response to c-di-AMP and 3'3' cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-? signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.
| S-EPMC3804601 | biostudies-literature
Project description:Activation of the stimulator of interferon genes (STING) pathway by microbial or self-DNA, as well as cyclic dinucleotides (CDNs), results in the induction of numerous genes that suppress pathogen replication and facilitate adaptive immunity. However, sustained gene transcription is rigidly prevented to avoid lethal STING-dependent proinflammatory disease by mechanisms that remain unknown. We demonstrate here that, after autophagy-dependent STING delivery of TANK-binding kinase 1 (TBK1) to endosomal/lysosomal compartments and activation of transcription factors interferon regulatory factor 3 (IRF3) and NF-?B, STING is subsequently phosphorylated by serine/threonine UNC-51-like kinase (ULK1/ATG1), and IRF3 function is suppressed. ULK1 activation occurred following disassociation from its repressor AMP activated protein kinase (AMPK) and was elicited by CDNs generated by the cGAMP synthase, cGAS. Thus, although CDNs may initially facilitate STING function, they subsequently trigger negative-feedback control of STING activity, thus preventing the persistent transcription of innate immune genes.
| S-EPMC3881181 | biostudies-other
Project description:Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.
| S-EPMC6446192 | biostudies-literature
Project description:The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage. Cytosolic DNA triggers immune responses by activating the cGAS/STING pathway. The binding of DNA to the cytosolic enzyme cGAMP synthase (cGAS), activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic[G(2’,5’)pA(3’,5’)] (2’3’-cGAMP). 2’3’-cGAMP, a cyclic dinucleotide (CDN), activates the protein ‘stimulator of interferon genes’ (STING), which in turn activates the transcription factors IRF3 and NF-κB promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2’3’-cGAMP produced by malignant cells and other CDNs, including CDNs produced by bacteria and synthetic CDNs used in cancer immunotherapy, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter for CDNs. CDN uptake and functional responses are inhibited by depleting SLC19A1 from human cells and enhanced by overexpressing SLC19A1. In both human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates, as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer, host responsiveness to CDN-producing pathogenic microorganisms, and potentially in certain inflammatory diseases.
2019-09-01 | GSE134371 | GEO
Project description:The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway3. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)4-7. This cyclic dinucleotide (CDN) activates STING8, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells9 and other CDNs, including those produced by bacteria10-12 and synthetic CDNs used in cancer immunotherapy13,14, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer13, host responsiveness to CDN-producing pathogenic microorganisms11 and-potentially-for some inflammatory diseases.
| S-EPMC6785039 | biostudies-literature
Project description:Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-β, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-β is also important for the UPR induction during B. abortus infection. Indeed, IFN-β shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-β favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-β signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.
| S-EPMC6478548 | biostudies-literature
Project description:UnlabelledCoronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as "non-self" by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.ImportanceThe innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described to date. The introduction of a mutation in zinc finger 1 of the ExoN domain of nsp14 led to production of a virus that induced a weak antiviral response, most likely due to the accumulation of lower levels of dsRNA in the late phases of infection. These observations allowed us to propose a novel role for CoV nsp14 ExoN activity in counteracting the antiviral response, which could serve as a novel target for the design of antiviral strategies.
| S-EPMC4934755 | biostudies-literature
Project description:SLC19A1 transports immunoreactive cyclic dinucleotides
| PRJNA554882 | ENA
Project description:Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.
| S-EPMC3518029 | biostudies-literature