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• W2967490285 abstract "Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Consequently, GRA15−/− T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner. Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Consequently, GRA15−/− T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner. T. gondii inveSTING in a latent futureJournal of Biological ChemistryVol. 294Issue 45PreviewToxoplasma gondii is an obligate protozoan parasite that naturally infects all mammals, where it alters the host environment to establish chronic infection. Wang and colleagues uncover a new role for the T. gondii protein GRA15 in inducing an anti-parasite response via the interferon stimulator STING. This parasite-driven host defense limits Toxoplasma replication while maintaining host survival, creating an ideal niche for the establishment of latency. Full-Text PDF Open Access The protozoan parasite Toxoplasma gondii can infect nearly all warm-blooded animals (1Robert-Gangneux F. Dardé M.L. Epidemiology of and diagnostic strategies for toxoplasmosis.Clin. Microbiol. Rev. 2012; 25 (22491772): 264-29610.1128/CMR.05013-11Crossref PubMed Scopus (1003) Google Scholar, 2Liu Q. Wang Z.D. Huang S.Y. Zhu X.Q. Diagnosis of toxoplasmosis and typing of Toxoplasma gondii.Parasit. Vectors. 2015; 8 (26017718): 29210.1186/s13071-015-0902-6Crossref PubMed Scopus (234) Google Scholar). As for humans, nearly 30% of the world's population is infected with T. gondii (3Montoya J.G. Liesenfeld O. Toxoplasmosis.Lancet. 2004; 363 (15194258): 1965-197610.1016/S0140-6736(04)16412-XAbstract Full Text Full Text PDF PubMed Scopus (2538) Google Scholar). In healthy adults, T. gondii is controlled by the immune system and remains dormant in the brain. However, in immunocompromised individuals, a defect of the immune system leads to the reactivation of the T. gondii parasites and the development of toxoplasmosis. Reactivated parasite replication causes life-threatening brain damage with brain abscesses and necrotic areas (4Yarovinsky F. Innate immunity to Toxoplasma gondii infection.Nat. Rev. Immunol. 2014; 14 (24457485): 109-12110.1038/nri3598Crossref PubMed Scopus (277) Google Scholar). Thus, HIV/AIDS patients, cancer patients, and organ transplant recipients are highly susceptible to T. gondii infection. The infection of T. gondii parasites is recognized by pattern recognition receptors (PRRs). 4The abbreviations used are: PRRpattern recognition receptorqRT-PCRquantitative RT-PCRWCLwhole-cell lysateERendoplasmic reticulumSDD-AGEsemidenaturing detergent–agarose gel electrophoresisDCdendritic cellPVparasitophorous vacuoleGRAgranuleIFNinterferonTRAFTNF receptor-associated factorDAPI4′,6-diamidino-2-phenylindoleISGinterferon-stimulated genecGAScyclic GMP-AMP synthaseiBMDMimmortalized bone marrow-derived macrophagePTMpost-translational modificationHFFhuman foreskin fibroblastDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumRNA-seqRNA-sequencingMEFmouse embryo fibroblastRLRRIG-I-like (retinoic acid-inducible gene I-like) receptorsMAVSmitochondrial antiviral-signaling protein. Previous studies showed that TLR11 is the PRR of T. gondii in murine cells. TLR11 is able to detect the actin-binding protein Profilin, which is required for entry of T. gondii during infection. TLR11 and TLR12 form a heterodimer in murine dendritic cells (DC) after sensing Profilin and activate adaptor protein MyD88 to initiate downstream signaling for defense against T. gondii (5Koblansky A.A. Jankovic D. Oh H. Hieny S. Sungnak W. Mathur R. Hayden M.S. Akira S. Sher A. Ghosh S. Recognition of profilin by Toll-like receptor 12 is critical for host resistance to Toxoplasma gondii.Immunity. 2013; 38 (23246311): 119-13010.1016/j.immuni.2012.09.016Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Moreover, TLR7 and TLR9 are able to compensate for the loss of TLR11 by activating MyD88 in TLR11-deficient mice (6Andrade W.A. Souza Mdo C. Ramos-Martinez E. Nagpal K. Dutra M.S. Melo M.B. Bartholomeu D.C. Ghosh S. Golenbock D.T. Gazzinelli R.T. Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice.Cell Host Microbe. 2013; 13 (23290966): 42-5310.1016/j.chom.2012.12.003Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Interestingly, TLR7 is activated by T. gondii RNA and triggers innate immune signaling only in TLR11-deficient cells (7Pisitkun P. Deane J.A. Difilippantonio M.J. Tarasenko T. Satterthwaite A.B. Bolland S. Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication.Science. 2006; 312 (16709748): 1669-167210.1126/science.1124978Crossref PubMed Scopus (690) Google Scholar). In addition, TLR7 expression is undetectable in CD9α + DCs, which is believed to be the primary DC subset for surveillance of the infection of T. gondii (8Edwards A.D. Diebold S.S. Slack E.M. Tomizawa H. Hemmi H. Kaisho T. Akira S. Reis e Sousa C. Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 α+ DC correlates with unresponsiveness to imidazoquinolines.Eur. J. Immunol. 2003; 33 (12672047): 827-83310.1002/eji.200323797Crossref PubMed Scopus (464) Google Scholar). Therefore, the importance of TLR7 in defense against T. gondii is unclear. TLR9 recognizes the methylated DNA of T. gondii (9Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. A Toll-like receptor recognizes bacterial DNA.Nature. 2000; 408 (11130078): 740-74510.1038/35047123Crossref PubMed Scopus (5360) Google Scholar, 10Gissot M. Choi S.W. Thompson R.F. Greally J.M. Kim K. Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation.Eukaryot. Cell. 2008; 7 (18178772): 537-54010.1128/EC.00448-07Crossref PubMed Scopus (47) Google Scholar). However, TLR9 expression is not always detected in the T. gondii–infected DC, whose expression needs priming by IFNγ (6Andrade W.A. Souza Mdo C. Ramos-Martinez E. Nagpal K. Dutra M.S. Melo M.B. Bartholomeu D.C. Ghosh S. Golenbock D.T. Gazzinelli R.T. Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice.Cell Host Microbe. 2013; 13 (23290966): 42-5310.1016/j.chom.2012.12.003Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition to those receptors localized in endosomes, the cell plasma membrane receptors TLR2 and TLR4 are shown to recognize the glycosylphosphatidylinositol from the surface of T. gondii and T. gondii-derived heat-shock protein 70 (HSP70) (11Debierre-Grockiego F. Campos M.A. Azzouz N. Schmidt J. Bieker U. Resende M.G. Mansur D.S. Weingart R. Schmidt R.R. Golenbock D.T. Gazzinelli R.T. Schwarz R.T. Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii.J. Immunol. 2007; 179 (17617606): 1129-113710.4049/jimmunol.179.2.1129Crossref PubMed Scopus (219) Google Scholar, 12Mun H.S. Aosai F. Norose K. Piao L.X. Fang H. Akira S. Yano A. Toll-like receptor 4 mediates tolerance in macrophages stimulated with Toxoplasma gondii-derived heat shock protein 70.Infect. Immun. 2005; 73 (16040976): 4634-464210.1128/IAI.73.8.4634-4642.2005Crossref PubMed Scopus (51) Google Scholar). However, some studies showed that T. gondii suppressed innate immune responses via TLR4. Leng and Denkers (13Leng J. Denkers E.Y. Toxoplasma gondii inhibits covalent modification of histone H3 at the IL-10 promoter in infected macrophages.PLoS One. 2009; 4 (19859561): e758910.1371/journal.pone.0007589Crossref PubMed Scopus (35) Google Scholar) found that chromatin remodeling in T. gondii–infected macrophages inhibited cytokine production via TLR4. Lee et al. (14Lee E.J. Heo Y.M. Choi J.H. Song H.O. Ryu J.S. Ahn M.H. Suppressed production of pro-inflammatory cytokines by LPS-activated macrophages after treatment with Toxoplasma gondii lysate.Korean J. Parasitol. 2008; 46 (18830053): 145-15110.3347/kjp.2008.46.3.145Crossref PubMed Scopus (14) Google Scholar) also reported that T. gondii suppressed the production of pro-inflammatory cytokines after TLR4–ligand interactions. The well-studied murine PRRs of T. gondii are TLR11 and TLR12. However, the human genome does not encode TLR11 or TLR12 proteins. Nearly all the known T. gondii PRRs are membrane-harbored on plasma membrane or endosome. Cytoplasmic PPR pathways are also crucial for sensing invading pathogens and initiating host defense. The cytoplasmic sensors for virus and bacterium have been well-characterized (15Delbridge L.M. O'Riordan M.X. Innate recognition of intracellular bacteria.Curr. Opin. Immunol. 2007; 19 (17126540): 10-1610.1016/j.coi.2006.11.005Crossref PubMed Scopus (117) Google Scholar16Akira S. Uematsu S. Takeuchi O. Pathogen recognition and innate immunity.Cell. 2006; 124 (16497588): 783-80110.1016/j.cell.2006.02.015Abstract Full Text Full Text PDF PubMed Scopus (8722) Google Scholar, 17Neyen C. Lemaitre B. Sensing Gram-negative bacteria: a phylogenetic perspective.Curr. Opin. Immunol. 2016; 38 (26569344): 8-1710.1016/j.coi.2015.10.007Crossref PubMed Scopus (43) Google Scholar18Wilkins C. Gale Jr., M. Recognition of viruses by cytoplasmic sensors.Curr. Opin. Immunol. 2010; 22 (20061127): 41-4710.1016/j.coi.2009.12.003Crossref PubMed Scopus (338) Google Scholar). Moreover, T. gondii is an intracellular pathogen. A previous study (19Ewald S.E. Chavarria-Smith J. Boothroyd J.C. NLRP1 is an inflammasome sensor for Toxoplasma gondii.Infect. Immun. 2014; 82 (24218483): 460-46810.1128/IAI.01170-13Crossref PubMed Scopus (166) Google Scholar) showed that NLRP1 is an inflammasome sensor for T. gondii, which influences susceptibility to human congenital toxoplasmosis (20Witola W.H. Mui E. Hargrave A. Liu S. Hypolite M. Montpetit A. Cavailles P. Bisanz C. Cesbron-Delauw M.F. Fournié G.J. McLeod R. NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells.Infect. Immun. 2011; 79 (21098108): 756-76610.1128/IAI.00898-10Crossref PubMed Scopus (138) Google Scholar). However, T. gondii was not able to activate NLRP1 signaling without pretreatment of lipopolysaccharide. Moreover, the inflammasome mainly mediated maturation of proinflammatory cytokines and cell pyroptosis but not induction of immune genes. Therefore, the PRRs of T. gondii, particularly the cytoplasmic pathogen-sensing pathway modulating induction of immune effectors, wait to be uncovered. pattern recognition receptor quantitative RT-PCR whole-cell lysate endoplasmic reticulum semidenaturing detergent–agarose gel electrophoresis dendritic cell parasitophorous vacuole granule interferon TNF receptor-associated factor 4′,6-diamidino-2-phenylindole interferon-stimulated gene cyclic GMP-AMP synthase immortalized bone marrow-derived macrophage post-translational modification human foreskin fibroblast Dulbecco's modified Eagle's medium fetal bovine serum RNA-sequencing mouse embryo fibroblast RIG-I-like (retinoic acid-inducible gene I-like) receptors mitochondrial antiviral-signaling protein. The classic antiviral cytokine interferon (IFN) β can be induced during parasitic infection. It has been shown that IFNβ was able to inhibit replication of T. gondii (21Schmitz J.L. Carlin J.M. Borden E.C. Byrne G.I. β-Interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages.Infect. Immun. 1989; 57 (2506136): 3254-3256Crossref PubMed Google Scholar, 22Orellana M.A. Suzuki Y. Araujo F. Remington J.S. Role of β-interferon in resistance to Toxoplasma gondii infection.Infect. Immun. 1991; 59 (1908831): 3287-3290Crossref PubMed Google Scholar). A recent study showed that a small group of atypical strains are able to induce type I IFN production in bone marrow-derived macrophages (BMDM) in a RIG-I- and IRF3-dependent manner. The canonical type I, II, or III strains failed to trigger type I IFN induction (23Melo M.B. Nguyen Q.P. Cordeiro C. Hassan M.A. Yang N. McKell R. Rosowski E.E. Julien L. Butty V. Dardé M.L. Ajzenberg D. Fitzgerald K. Young L.H. Saeij J.P. Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways.PLoS Pathog. 2013; 9 (24367253): e100377910.1371/journal.ppat.1003779Crossref PubMed Scopus (66) Google Scholar). However, Robey and co-workers (24Han S.J. Melichar H.J. Coombes J.L. Chan S.W. Koshy A.A. Boothroyd J.C. Barton G.M. Robey E.A. Internalization and TLR-dependent type I interferon production by monocytes in response to Toxoplasma gondii.Immunol. Cell Biol. 2014; 92 (25155465): 872-88110.1038/icb.2014.70Crossref PubMed Scopus (32) Google Scholar) found inflammatory monocytes produce IFNβ in response to type II T. gondii infection, suggesting different types of cells respond to T. gondii infection differently. cGMP-AMP synthase (cGAS) is the cytoplasmic double-stranded DNA (dsDNA) sensor recognizing foreign dsDNA and abnormal host dsRNA, which is critical for defending against DNA viruses and regulating homeostasis of immune system (25Cai X. Chiu Y.H. Chen Z.J. The cGAS–cGAMP–STING pathway of cytosolic DNA sensing and signaling.Mol. Cell. 2014; 54 (24766893): 289-29610.1016/j.molcel.2014.03.040Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 26Chen Q. Sun L. Chen Z.J. Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing.Nat. Immunol. 2016; 17 (27648547): 1142-114910.1038/ni.3558Crossref PubMed Scopus (953) Google Scholar). cGAS synthesizes secondary messenger cGAMP, which in turn activates STING (stimulator of interferon genes protein). STING is a key signal molecule localized in ER. Activated STING recruits kinase TBK1 that phosphorylates IRF3/IRF7 to initiate transcription of type I IFNs. It is believed that post-translational modification of STING modulates its activity and protein stability. TBK1 phosphorylates STING at serine 366, which is critical for IRF3 recruitment and type I IFN induction (27Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. Du F. Ren J. Wu Y.T. Grishin N.V. Chen Z.J. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347 (25636800): aaa263010.1126/science.aaa2630Crossref PubMed Scopus (941) Google Scholar). E3 ligase TRIM56 is able to attach Lys-63–linked polyubiquitin chain to STING and promotes dimerization and activation of STING (28Tsuchida T. Zou J. Saitoh T. Kumar H. Abe T. Matsuura Y. Kawai T. Akira S. The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular double-stranded DNA.Immunity. 2010; 33 (21074459): 765-77610.1016/j.immuni.2010.10.013Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). E3 protein complex autocrine motility factor receptor and insulin-induced gene 1 (INSIG1) promote Lys-27–linked polyubiquitination of STING and direct its translocation from the ER to the perinuclear vesicle (29Wang Q. Liu X. Cui Y. Tang Y. Chen W. Li S. Yu H. Pan Y. Wang C. The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING.Immunity. 2014; 41 (25526307): 919-93310.1016/j.immuni.2014.11.011Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). RNF5 ubiquitinates STING at lysine 150 resulting in its degradation by the proteasome, which could be antagonized by RNF26 that attaches Lys-11-linked polyubiquitin to the same lysine of STING (30Qin Y. Zhou M.T. Hu M.M. Hu Y.H. Zhang J. Guo L. Zhong B. Shu H.B. RNF26 temporally regulates virus-triggered type I interferon induction by two distinct mechanisms.PLoS Pathog. 2014; 10 (25254379): e100435810.1371/journal.ppat.1004358Crossref PubMed Scopus (129) Google Scholar, 31Zhong B. Zhang L. Lei C. Li Y. Mao A.P. Yang Y. Wang Y.Y. Zhang X.L. Shu H.B. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.Immunity. 2009; 30 (19285439): 397-40710.1016/j.immuni.2009.01.008Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). However, the role of cGAS/STING in the host defense against T. gondii has not been well-characterized. Accumulating studies have shown that T. gondii virulence is mediated by effector proteins secreted by parasites into the host cell. Dense granule proteins (GRAs) are produced by T. gondii, many of which are secreted into the parasitophorous vacuole (PV) and contribute to the maturation and structural integrity of the PV (32Rommereim L.M. Bellini V. Fox B.A. Pètre G. Rak C. Touquet B. Aldebert D. Dubremetz J.F. Cesbron-Delauw M.F. Mercier C. Bzik D.J. Phenotypes associated with knockouts of eight dense granule gene loci (GRA2-9) in virulent Toxoplasma gondii.PLoS One. 2016; 11 (27458822): e015930610.1371/journal.pone.0159306Crossref PubMed Scopus (27) Google Scholar). GRA proteins also participate in the modulation of host defense during infection (33Hiszczyńska-Sawicka E. Olędzka G. Holec-Gąsior L. Li H. Xu J.B. Sedcole R. Kur J. Bickerstaffe R. Stankiewicz M. Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6, and GRA7 antigens of Toxoplasma gondii.Vet. Parasitol. 2011; 177 (21251760): 281-28910.1016/j.vetpar.2010.11.047Crossref PubMed Scopus (58) Google Scholar, 34Nam H.W. GRA proteins of Toxoplasma gondii: maintenance of host–parasite interactions across the parasitophorous vacuolar membrane.Korean J. Parasitol. 2009; 47 (19885333): S29-S3710.3347/kjp.2009.47.S.S29Crossref PubMed Scopus (75) Google Scholar). GRA proteins mainly suppress host immune responses and facilitate parasite replication (35Hunter C.A. Sibley L.D. Modulation of innate immunity by Toxoplasma gondii virulence effectors.Nat. Rev. Microbiol. 2012; 10 (23070557): 766-77810.1038/nrmicro2858Crossref PubMed Scopus (358) Google Scholar). However, GRA15 activates NF-κB signaling in a TRAF6-dependent way, and in turn it up-regulates the production of IL12 (36Rosowski E.E. Lu D. Julien L. Rodda L. Gaiser R.A. Jensen K.D. Saeij J.P. Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein.J. Exp. Med. 2011; 208 (21199955): 195-21210.1084/jem.20100717Crossref PubMed Scopus (294) Google Scholar). Moreover, GRA15 promotes the secretion of IL1β via inflammatory monocytes during infection (37Gov L. Karimzadeh A. Ueno N. Lodoen M.B. Human innate immunity to Toxoplasma gondii is mediated by host caspase-1 and ASC and parasite GRA15.MBio. 2013; 4 (23839215): e00213-e0025510.1128/mBio.00255-13Crossref Scopus (83) Google Scholar). In this study, we found that cGAS is required for innate immune response against T. gondii. cGAS-deficient mice are much more susceptible to lethal infection of T. gondii than WT mice. Loss of STING results in a more severe phenotype during T. gondii infection. Mechanistically, we demonstrated that secreted dense granule protein GRA15 promotes oligomerization and activation of STING. GRA15 promotes polyubiquitination of STING at lysine 337 via TRAF proteins, which is required for activation of STING. Together, GRA15 enhances cGAS/STING signaling by potentiating STING activity during T. gondii infection. The cGAS/STING axis recognizes cytoplasmic DNA and triggers innate immune responses. Here, we investigated the role of STING in host defense against the infection of T. gondii. The STING loss–of–function Tmem173gt/gt mice (also known as Golden ticket and here referred to Stinggt/gt) (38Sauer J.D. Sotelo-Troha K. von Moltke J. Monroe K.M. Rae C.S. Brubaker S.W. Hyodo M. Hayakawa Y. Woodward J.J. Portnoy D.A. Vance R.E. The N-ethyl-N-nitrosourea–induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides.Infect. Immun. 2011; 79 (21098106): 688-69410.1128/IAI.00999-10Crossref PubMed Scopus (394) Google Scholar) and WT mice were infected with type II T. gondii (ME49 strain). Interestingly, Stinggt/gt mice were more susceptible to T. gondii infection than WT animals (Fig. 1A). Consistent with the survival results, the parasite burden (as assessed by T. gondii B1 DNA) was significantly higher in the spleen of Stinggt/gt mice than WT mice after T. gondii infection (Fig. 1B). Furthermore, we detected less type I IFNs, including IFNβ and IFNa4 in the spleen of Stinggt/gt mice than WT mice during infection (Fig. 1C and Fig. S1A). Consequently, induction of interferon-stimulated genes, including ISG15 and CXCL10, was significantly impaired in STING mutant mice. In addition to type I IFNs, STING is also required for the induction of inflammatory cytokine interleukin 12 (IL12), which is believed critical for controlling T. gondii infection (Fig. 1C). Consistently, the induction of IFNγ is modestly decreased in Stinggt/gt mice (Fig. 1C). Therefore, STING is required for host defense against infection with T. gondii in vivo. At the cellular level, we isolated peritoneal macrophages from WT and Stinggt/gt mice and infected these cells with T. gondii. In line with the animal experiments, macrophages with mutant STING failed to mount production of IFNβ, ISG15, CXCL10, and IL12 during T. gondii infection (Fig. 1D). To further confirm these findings, we infected immortalized Bone Marrow-Derived Macrophages (iBMDM) with T. gondii after deleting endogenous STING by using CRISPR/Cas9. The lacking of STING resulted in more parasite replication in iBMDM, which was measured by immunofluorescence and T. gondii B1 DNA qRT-PCR (Fig. 1E and Fig. S1B). The induction of ISGs and IL12 was also significantly decreased in STING-deficient iBMDM, although the induction of these cytokines in WT iBMDM was not as robust as that in WT primary peritoneal macrophages (Fig. 1F). Interestingly, we did not observe the induction of IFNβ by T. gondii infection in this cell type (Fig. S1C). This could be due to the specificity of cell type–dependent pathways or immune evasion strategies utilized by T. gondii. As cGAS is a cytoplasmic DNA sensor that functions upstream of STING (39Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339 (23258413): 786-79110.1126/science.1232458Crossref PubMed Scopus (2508) Google Scholar), we reasoned that cGAS might also be involved in anti-T. gondii responses by sensing the cytoplasmic DNA. We then evaluated the involvement of cGAS in anti-T. gondii immune responses. The mouse survival experiment showed that cGAS was required for anti-T. gondii responses (Fig. 1G). In addition to DNA, RNA from the invading pathogen can also trigger innate immunity of the host. RLRs recognize foreign RNA in the cytoplasm and activate adaptor protein MAVS. To this end, we infected MAVS-deficient mice, whose cytoplasmic RNA-sensing pathway was blocked, or WT mice with T. gondii. The survival and body weight results showed that MAVS was dispensable for anti-T. gondii immune responses (Fig. S1D). Together, cGAS and STING are crucial for innate immunity against T. gondii. During T. gondii infection in cGAS-deficient mice, interestingly, we noticed that loss of cGAS resulted in less severe symptoms and delayed death than STING deficiency, indicating an additional mechanism contributing to STING-mediated defense exists. We next focused on revealing this additional mechanism by which T. gondii activates STING signaling. In addition to nucleic acids, proteins from pathogens are also able to regulate host immune responses (35Hunter C.A. Sibley L.D. Modulation of innate immunity by Toxoplasma gondii virulence effectors.Nat. Rev. Microbiol. 2012; 10 (23070557): 766-77810.1038/nrmicro2858Crossref PubMed Scopus (358) Google Scholar, 40Melo M.B. Jensen K.D. Saeij J.P. Toxoplasma gondii effectors are master regulators of the inflammatory response.Trends Parasitol. 2011; 27 (21893432): 487-49510.1016/j.pt.2011.08.001Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). It is believed that the effector proteins secreted by T. gondii are able to modulate host immune responses (41Hakimi M.A. Bougdour A. Toxoplasma's ways of manipulating the host transcriptome via secreted effectors.Curr. Opin. Microbiol. 2015; 26 (25912924): 24-3110.1016/j.mib.2015.04.003Crossref PubMed Scopus (56) Google Scholar). Dense granule proteins (GRAs) have emerged as important determinants of T. gondii pathogenesis. To determine whether the T. gondii proteins affect innate immune activation, we constructed plasmids encoding various dense granule proteins of T. gondii and transfected these plasmids together with cGAS, STING, and IFNβ luciferase reporter plasmids. We found that only GRA15, which was reported to regulate NF-κB activation (36Rosowski E.E. Lu D. Julien L. Rodda L. Gaiser R.A. Jensen K.D. Saeij J.P. Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein.J. Exp. Med. 2011; 208 (21199955): 195-21210.1084/jem.20100717Crossref PubMed Scopus (294) Google Scholar), was able to significantly enhance cGAS/STING-mediated induction of IFNβ (Fig. 2A). We then investigated the effect of GRA15 on parasite virulence. We generated GRA15-deficient T. gondii by using CRISPR/Cas9 (Fig. 2B). In contrast to the previous finding (36Rosowski E.E. Lu D. Julien L. Rodda L. Gaiser R.A. Jensen K.D. Saeij J.P. Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein.J. Exp. Med. 2011; 208 (21199955): 195-21210.1084/jem.20100717Crossref PubMed Scopus (294) Google Scholar), C57BL/6 mice were more susceptible to GRA15−/− T. gondii than WT T. gondii (Fig. 2C). Consistent with the survival data, mice infected with GRA15−/− T. gondii showed significantly higher parasite burden in the spleen than mice infected with WT T. gondii (Fig. 2D). Moreover, replication of GRA15−/− T. gondii significantly increased in MEFs compared with WT parasites (Fig. S2A). Furthermore, GRA15−/− T. gondii induced less IFNβ, ISG15, CXCL10, IFNγ, and IL12 than WT T. gondii did in the spleen (Fig. 2E), although the decrease of IFNβ induction was not as significant as other cytokines. Consequently, GRA15−/− parasite infection caused more severe mortality of mice. These results suggested that GRA15 is required for competent induction of innate immune responses. To further confirm this, we infected iBMDM with WT or GRA15−/− T. gondii. qRT-PCR assay showed that the production of cytokines was severely impaired during GRA15−/− T. gondii infection compared with WT parasite infection (Fig. 2F). To examine whether GRA15 is involved in STING signaling in vivo, we infected Stinggt/gt mice with WT or GRA15−/− parasite. Th" @default.
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• W2967490285 date "2019-11-01" @default.
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• W2967490285 title "The GRA15 protein from Toxoplasma gondii enhances host defense responses by activating the interferon stimulator STING" @default.
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