{"gene":"TG_203310","gene_symbol":"GRA7","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2006,"finding":"GRA7 acts as a tubulogenic protein that decorates microtubule-based invaginations of the parasitophorous vacuole membrane (PVM), functioning like a garrote to sequester host endo-lysosomes within the vacuolar space. The parasite actively recruits host microtubules to the PV, and these tubular conduits decorated by GRA7 mediate unidirectional transport and sequestration of host endocytic organelles.","method":"Immunoelectron microscopy, fluorescence microscopy, subcellular fractionation, live imaging of host endo-lysosome recruitment to PV","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct imaging and fractionation demonstrating GRA7 decoration of tubular conduits and endo-lysosome sequestration, published in high-impact journal with multiple orthogonal methods","pmids":["16630815"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"GRA7 (p29) is a dense granule protein that, after host cell invasion, is secreted into the parasitophorous vacuole (PV) and associates with the PV membrane and intravacuolar network. In bradyzoite-infected cells, GRA7 is present in the host cell cytoplasm and is released into culture supernatant, suggesting excretion from cyst-bearing cells. The protein contains a putative N-terminal signal peptide and a C-terminal hydrophobic transmembrane domain, and is expressed in all three intraspecies subgroups.","method":"Immunoelectron microscopy, immunofluorescence, Northern blot, ELISA, protein sequencing from tryptic peptides, genomic DNA amplification","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent papers (PMID 9566518 and 9566517) using multiple orthogonal methods (immunoelectron microscopy, immunofluorescence, biochemistry) establishing dense granule localization and post-invasion secretion","pmids":["9566518","9566517"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"BK"}]},{"year":1998,"finding":"Ultrastructural immunolocalization demonstrated that GRA7 (P29) traffics from dense granules via the intravacuolar network to the PVM after tachyzoite invasion, associating with the parasite membrane complex, tubular elements of the intravacuolar network, and the parasitophorous vacuolar membrane.","method":"Immunogold electron microscopy on thin sections using monoclonal antibody 5-241-178","journal":"The journal of histochemistry and cytochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — ultrastructural immunolocalization with quantitative immunogold, single lab but rigorous method establishing trafficking route","pmids":["9815283"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"GRA7 is exclusively membrane-associated in parasites and infected host cells. Triton X-114 partitioning demonstrates GRA7 is an integral membrane protein, with the hydrophobic stretch from amino acids 181–202 serving as a membrane anchor. GRA7 is detected on the surface of intact host cells (originating intracellularly), and additional slower-migrating modified forms of GRA7 appear in membranous fractions from infected host cells, indicating post-exocytosis modification. GRA7 expression level correlates with T. gondii virulence, with a massive decrease in GRA7 synthesis in an in vitro-attenuated variant. GRA7-specific antibodies are present in cerebrospinal fluid during chronic infection, consistent with a role at the parasite-host interface.","method":"Proteome analysis, immunoblot, subcellular fractionation, Triton X-114 partitioning, flow cytometry","journal":"Microbes and infection","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods (fractionation, detergent partitioning, flow cytometry) in a single lab establishing integral membrane character and host cell surface localization","pmids":["12048027"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"BK"}]},{"year":1999,"finding":"GRA7 is expressed in all infectious life cycle stages of T. gondii (tachyzoite, bradyzoite, merozoite, sporozoite) and is uniquely the only dense granule protein also expressed in enteric (coccidian) stages in the cat gut. During tachyzoite development, GRA7 is secreted and associates with the parasitophorous vacuole. In bradyzoites within mature cysts, GRA7 staining of the cyst wall is reduced compared to tachyzoites. In enteric forms, GRA7 strongly labels PV-containing early asexual and sexual stages. This stage-specific pattern differentiates GRA7 from GRA1–6.","method":"Immunocytochemistry with stage-specific markers (SAG1, BAG1), electron microscopy, in vivo infection model (cat and intermediate host)","journal":"Parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo immunocytochemistry with double labeling using established stage markers, single lab","pmids":["10503251"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"GRA proteins, including GRA7, form high-molecular-weight oligomeric complexes within the parasite dense granules and within the PV soluble fraction. Affinity purification of HA-FLAG-tagged GRA2 and GRA5 revealed that GRA3, GRA6, and GRA7 are major partners in these complexes. After insertion into PV membranes, GRA2 associates with GRA5 and GRA7, while GRA5 associates with GRA7 only, suggesting progressive loss of partners upon membrane insertion.","method":"Affinity chromatography (HA-FLAG tag purification) of knockout parasites complemented with tagged GRA2 or GRA5, conventional chromatography, immunoblot","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — affinity purification from engineered knockout-complemented parasites with multiple GRA partners identified; rigorous genetic background with reciprocal pulls","pmids":["17959262"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"GRA7 is phosphorylated in infected host cells but not in extracellular parasites, indicating host cell-dependent phosphorylation. Within 10 min of invasion, GRA7 appears in strand-like structures in the host cytosol that also contain rhoptry proteins. Independently of its phosphorylation state, GRA7 associates with rhoptry proteins ROP2 and ROP4 in infected host cells. GRA7 strands also contain the dense granule proteins GRA1 and GRA3.","method":"Immunoprecipitation, immunofluorescence, phosphorylation analysis of extracellular vs. intracellular parasites, co-localization studies","journal":"Infection and immunity","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-immunoprecipitation demonstrating GRA7-ROP2/ROP4 association, phosphorylation established by comparing intra- vs. extracellular conditions, multiple orthogonal methods","pmids":["18809661"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"GRA7 exists in a complex with the pseudokinases ROP8/ROP2 and the active kinase ROP18 at the PVM. Individual deletion of gra7 or rop18 partially attenuates virulence in mice, while the double gra7/rop18 mutant is avirulent, placing GRA7 and ROP18 in the same pathway. GRA7 directly binds the active GTP-bound dimer of immunity-related GTPase a6 (Irga6) in a GTP-dependent manner, leading to enhanced Irga6 polymerization, rapid turnover, and eventual disassembly. Increased IRG recruitment and parasite clearance occur in IFN-γ-activated macrophages lacking GRA7 and ROP18.","method":"Genetic deletion (single and double knockout), in vivo virulence assays in mice, direct binding assay (GRA7 to Irga6 dimer), IFN-γ-activated macrophage clearance assay, epistasis analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — combination of genetic epistasis (double KO avirulent), direct in vitro binding assay, and functional macrophage clearance assay; multiple orthogonal methods in one rigorous study","pmids":["24390541"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2015,"finding":"GRA7 is an additional component of the ROP5/ROP18 kinase complex and associates with the PVM through direct binding to ROP5. Absence of GRA7 results in reduced phosphorylation of Irga6, increased vacuolar IRG protein amounts, and attenuated virulence. GRA7 is strictly an Irga6-specific virulence effector (not targeting other IRG family members), functioning as a regulator for ROP18-specific inactivation of Irga6.","method":"Co-immunoprecipitation, genetic deletion analysis, Irga6 phosphorylation assay, in vivo virulence assay, IRG recruitment quantification","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP establishing GRA7-ROP5 interaction, genetic deletion with Irga6 phosphorylation readout, replication of GRA7-IRG axis from PMID 24390541","pmids":["26247512"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"GRA7 induces MyD88-dependent signaling through TRAF6 activation and ROS production, leading to NF-κB-mediated proinflammatory responses in macrophages. GRA7 physically associates with TRAF6 via MyD88. The C-terminal domain of GRA7 (GRA7-V) is sufficient for interaction with and ubiquitination of the RING domain of TRAF6, enabling inflammatory cytokine production. PKCα-independent ROS generation and TRAF6 activation are mutually dependent on GRA7/MyD88 signaling.","method":"Co-immunoprecipitation (GRA7-TRAF6 interaction), domain-deletion mapping (GRA7-V), ubiquitination assay, cytokine measurement, MyD88-knockout macrophages","journal":"Infection and immunity","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP plus domain mapping plus functional ubiquitination assay in a single study with multiple orthogonal methods","pmids":["26553469"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"PKCα-mediated phosphorylation of GRA7 at Ser52 (GRA7-I domain) regulates GRA7 interaction with the PYD domain of ASC (apoptosis-associated speck-like protein), enabling oligomerization and inflammasome activation for antimicrobial defense against Mycobacterium tuberculosis. GRA7-III domain interacts with the PX domain of phospholipase D1 (PLD1) in a Ser135 phosphorylation-dependent manner (also via PKCα), facilitating PLD1 enzyme activity and phago-lysosomal maturation, contributing to antimicrobial activity.","method":"Co-immunoprecipitation (GRA7-ASC, GRA7-PLD1), phosphorylation-site mutagenesis (Ser52, Ser135), PLD1 enzyme activity assay, phago-lysosomal maturation assay, inflammasome activation assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro binding with mutagenesis of phosphorylation sites, enzyme activity assay, and functional phagolysosomal readout; multiple orthogonal methods in one study","pmids":["28125719"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"Deletion of PVM-associated GRA7 (along with GRA3, GRA8, GRA14) in the type II Prugniaud strain induces severe defects in chronic cyst development in vivo without affecting in vitro growth rate or in vitro differentiation. GRA7 deletion parasites show reduced acute virulence. GRA7 (along with ROP5, ROP18) suppresses MHC-I antigen presentation of PV-localized parasite antigens to CD8+ T cells, with suppression correlated to prevention of PV disruption by IFN-γ-activated host IRG effectors.","method":"Genetic deletion in type II Pru strain, in vivo chronic infection model (mouse), in vitro differentiation assay, MHC-I antigen presentation assay, IRG recruitment assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic deletion with in vivo cyst formation, multiple GRA deletions compared, functional antigen presentation assay; replicated across two papers (PMID 31266861 and 31555296)","pmids":["31266861","31555296"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud; RH"}]},{"year":2020,"finding":"GRA7 and GRA14 (but not GRA15 alone) are partially involved in NF-κB activation; all three (GRA7, GRA14, GRA15) contribute to nuclear translocation of RelA. Deletion of GRA7, GRA14, and GRA15 decreases IL-6 levels in macrophages and downregulates NF-κB-mediated gene expression. Luciferase reporter assays confirm GRA7 overexpression activates NF-κB-dependent transcription. Deletion of GRA7 in the type II Pru strain increases parasite virulence, suggesting GRA7-induced host NF-κB immunity limits parasite expansion.","method":"Luciferase reporter assay (NF-κB), genetic deletion (Δgra7, Δgra14, Δgra15), RelA nuclear translocation assay, RNA-seq, cytokine measurement, in vivo virulence assay","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — luciferase reporter plus genetic deletion plus RNA-seq plus in vivo virulence; multiple orthogonal methods in one study","pmids":["32849602"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud"}]},{"year":2020,"finding":"GRA5 and GRA7 localize to the cyst periphery as early as 6 h after in vitro differentiation and remain at the cyst membrane and cyst wall region throughout cyst maturation (up to day 10), supporting the model that the PVM remains intact and develops into the cyst membrane. In mature cysts, saponin permeabilization selectively removes GRA5 but not GRA7 from the cyst membrane. Deletion of PVM-localized GRAs including GRA7 reduces the rate of cyst wall protein accumulation at the cyst periphery.","method":"Live imaging and immunofluorescence of GRA5/GRA7 during in vitro differentiation, saponin permeabilization assay, genetic deletion of individual GRAs with cyst wall protein accumulation readout","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Moderate — time-course live imaging plus biochemical permeabilization plus genetic deletion with functional cyst wall readout; multiple methods in one study","pmids":["31941814"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud"}]},{"year":2004,"finding":"GRA7 is expressed in tachyzoites, bradyzoites, and coccidian/enteric stages (merozoites), whereas GRA1–6 are absent in merozoites and enteric stages. In tachyzoites, GRA7 extensively labels the PV; in bradyzoites within cysts, the cyst wall shows variable staining. This unique expression in all stages is reflected in the differences in PV structure observed by electron microscopy across life cycle stages.","method":"Immunocytochemistry with panel of molecular markers, electron microscopy, in vivo infections in cat and intermediate host","journal":"International journal for parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo immunocytochemistry with multiple markers across life stages, corroborates PMID 10503251","pmids":["15003495"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"GRA7 protein is packaged into extracellular vesicles (EVs) released from Toxoplasma-infected neurons, and GRA7 can be observed within intracellular EVs and in the nuclei of GRA7+ EV-containing astrocytes after incubation with infected-neuron EVs. EVs from infected neurons altered astrocyte gene expression toward pro-inflammatory signatures and downregulated GLT-1 glutamate transporter expression.","method":"LC-MS/MS proteomic characterization of EVs, immunofluorescence (GRA7 in astrocyte nuclei), nanoparticle tracking analysis, transmission electron microscopy, RNA-seq of astrocytes","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — proteomic identification plus immunofluorescence in astrocytes, single lab, novel finding about GRA7 in EVs and nuclear translocation not yet independently replicated","pmids":["40523037"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"GRA60, a novel dense granule effector at the vacuolar space and PVM, contributes to resistance to IRG recruitment independently of GRA7. GRA60 is found associated with ROP18. Deletion of GRA60 in an RHΔrop5 background leads to IRG recruitment comparable to single knockouts, demonstrating GRA60 and ROP5 act in the same pathway. This study contextualizes GRA7 as one member of a broader virulence complex (GRA7, ROP5, ROP17, ROP18) resisting IRG-mediated PVM disruption.","method":"Genetic deletion of GRA60, IRG recruitment assay, co-immunoprecipitation (GRA60-ROP18), in vivo virulence assay","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GRA7 role confirmed as part of IRG-resistance complex; GRA60 data is primary finding but GRA7's role in the complex is corroborated","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"GRA7 is a dense granule protein expressed across all T. gondii life stages that is secreted after invasion into the parasitophorous vacuole (PV), where it integrates as an integral membrane protein into the PV membrane (PVM), intravacuolar network tubules, and cyst membrane; it acts as a tubulogenic garrote to sequester host endo-lysosomes within the vacuolar space, forms oligomeric complexes with GRA2, GRA3, GRA5, and GRA6, associates with rhoptry proteins ROP2, ROP4, ROP5, and ROP18 in a virulence complex that directly binds and disassembles the GTP-loaded Irga6 dimer to protect the PVM from IRG-mediated destruction and suppress MHC-I antigen presentation, activates host NF-κB and TRAF6/MyD88 innate immune signaling through its C-terminal domain, undergoes PKCα-mediated phosphorylation that controls interactions with ASC (inflammasome) and phospholipase D1 (phago-lysosomal maturation), and is required for normal cyst wall maturation during chronic infection."},"narrative":{"mechanistic_narrative":"GRA7 is a Toxoplasma gondii dense granule protein secreted after host cell invasion into the parasitophorous vacuole (PV), where it traffics via the intravacuolar network to the PV membrane (PVM) and integrates as an integral membrane protein through a hydrophobic membrane anchor (residues 181–202) [PMID:9566518, PMID:9566517, PMID:9815283, PMID:12048027]. It is expressed across all infectious life stages, uniquely including the enteric coccidian stages that lack other dense granule proteins, and persists at the cyst periphery during chronic infection [PMID:10503251, PMID:15003495, PMID:31941814]. At the PVM, GRA7 decorates microtubule-based tubular invaginations and acts as a tubulogenic \"garrote\" that sequesters host endo-lysosomes within the vacuolar space [PMID:16630815], and it assembles into high-molecular-weight oligomeric complexes with other GRA proteins including GRA2, GRA3, GRA5, and GRA6 [PMID:17959262]. GRA7 is a central effector of a virulence complex with the rhoptry proteins ROP2/ROP4, ROP5, and the active kinase ROP18: it associates with the PVM through direct binding to ROP5 and directly binds the GTP-loaded Irga6 dimer in a GTP-dependent manner, driving Irga6 phosphorylation, polymerization, and disassembly to protect the PVM from IRG-mediated destruction [PMID:18809661, PMID:24390541, PMID:26247512]. Genetic epistasis places GRA7 and ROP18 in the same pathway, and loss of GRA7 (with ROP5/ROP18) permits IRG recruitment, PV disruption, and increased MHC-I presentation of parasite antigens to CD8+ T cells [PMID:24390541, PMID:31266861, PMID:31555296]. GRA7 also engages host innate immune signaling, activating MyD88/TRAF6-dependent NF-κB proinflammatory responses through its C-terminal domain [PMID:26553469, PMID:32849602], and undergoes PKCα-mediated phosphorylation (Ser52, Ser135) that governs its interactions with ASC and phospholipase D1 in antimicrobial defense [PMID:28125719]. GRA7 is required for normal chronic cyst wall maturation in vivo [PMID:31266861, PMID:31555296, PMID:31941814].","teleology":[{"year":1998,"claim":"Establishing GRA7 as a secreted dense granule protein answered where it acts, defining its localization at the PV membrane and intravacuolar network after invasion.","evidence":"Immunoelectron microscopy, immunofluorescence, and biochemistry on tachyzoite- and bradyzoite-infected cells, plus immunogold trafficking analysis","pmids":["9566518","9566517","9815283"],"confidence":"High","gaps":["Membrane topology and mode of PVM insertion not yet resolved","Functional consequence of secretion not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":"BK"},{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"Documenting GRA7 expression across all life-cycle and enteric stages distinguished it from GRA1–6 and implicated it in every infectious form.","evidence":"Immunocytochemistry with stage-specific markers and electron microscopy in cat and intermediate-host infections","pmids":["10503251"],"confidence":"Medium","gaps":["Stage-specific function not defined","Reduced cyst-wall staining in bradyzoites left unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"Biochemical characterization resolved GRA7 as an integral membrane protein with a defined anchor and linked its expression to parasite virulence.","evidence":"Triton X-114 partitioning, subcellular fractionation, and flow cytometry, plus correlation with an attenuated variant","pmids":["12048027"],"confidence":"High","gaps":["Nature of post-exocytosis modifications not identified","Mechanistic basis of the virulence correlation not established"],"attribution":[{"species":"Toxoplasma gondii","strain":"BK"}]},{"year":2004,"claim":"Confirmation of GRA7 expression in tachyzoites, bradyzoites, and merozoites reinforced its unique pan-stage distribution relative to other GRAs.","evidence":"Immunocytochemistry with marker panel, electron microscopy, and in vivo infections","pmids":["15003495"],"confidence":"Medium","gaps":["Does not test function in any stage","Cyst-wall staining variability unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"claim":"Discovery of GRA7-decorated microtubule tubules answered how the PV manipulates host endocytic organelles, defining a tubulogenic sequestration role.","evidence":"Immunoelectron and fluorescence microscopy, fractionation, and live imaging of host endo-lysosome recruitment to the PV","pmids":["16630815"],"confidence":"High","gaps":["Molecular mechanism of tubule formation by GRA7 not defined","Contribution of sequestration to nutrient acquisition not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"claim":"Affinity purification defined GRA7 as a member of oligomeric GRA complexes, showing how dense granule proteins assemble before and after membrane insertion.","evidence":"HA-FLAG affinity purification from knockout-complemented parasites with reciprocal pulls and immunoblot","pmids":["17959262"],"confidence":"High","gaps":["Stoichiometry and structural arrangement of complexes unknown","Functional role of GRA7 within these complexes not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Detection of host-dependent phosphorylation and rhoptry-protein association linked GRA7 to ROP2/ROP4 in host-cytosolic strands, foreshadowing its virulence-complex role.","evidence":"Immunoprecipitation, immunofluorescence colocalization, and comparison of extracellular vs. intracellular parasite phosphorylation","pmids":["18809661"],"confidence":"High","gaps":["Kinase responsible for phosphorylation not identified here","Functional significance of GRA7-ROP2/ROP4 strands not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Genetic epistasis and direct binding assays revealed GRA7's core effector mechanism: disabling the host Irga6 GTPase to protect the PVM.","evidence":"Single and double gra7/rop18 knockouts, in vivo virulence assays, direct GRA7–Irga6 dimer binding assay, and IFN-γ macrophage clearance in RH strain","pmids":["24390541"],"confidence":"High","gaps":["Structural basis of GRA7–Irga6 recognition not resolved","Whether GRA7 targets IRGs beyond Irga6 not addressed here"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2015,"claim":"Defining GRA7 as a ROP5-binding component of the ROP5/ROP18 kinase complex answered how GRA7 is recruited to the PVM and regulates Irga6-specific inactivation.","evidence":"Reciprocal co-IP, genetic deletion, Irga6 phosphorylation and IRG recruitment quantification, and in vivo virulence","pmids":["26247512"],"confidence":"High","gaps":["Why GRA7 is Irga6-specific and not broader across IRGs not explained mechanistically","Strain background of this study unspecified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Mapping the GRA7 C-terminal domain to TRAF6 defined a distinct role activating host MyD88/NF-κB proinflammatory signaling.","evidence":"Co-IP, GRA7-V domain-deletion mapping, TRAF6 ubiquitination assay, and cytokine measurement in MyD88-knockout macrophages","pmids":["26553469"],"confidence":"High","gaps":["How PVM-integral GRA7 accesses cytosolic TRAF6 not resolved","In vivo relevance of the TRAF6 axis not established here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Identification of PKCα phosphosites (Ser52, Ser135) governing GRA7 binding to ASC and PLD1 connected GRA7 phosphorylation to inflammasome and phago-lysosomal antimicrobial defense.","evidence":"Co-IP, phosphosite mutagenesis, PLD1 enzyme activity, phago-lysosomal maturation, and inflammasome activation assays","pmids":["28125719"],"confidence":"High","gaps":["Whether these host-defense functions operate during Toxoplasma infection itself vs. heterologous contexts not delineated","Physiological trigger for PKCα phosphorylation not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Deletion studies in the type II Pru strain established GRA7's requirement for chronic cyst development and its role in suppressing MHC-I antigen presentation.","evidence":"Genetic deletion of PVM-associated GRAs, in vivo chronic infection, MHC-I presentation and IRG recruitment assays in Pru and RH","pmids":["31266861","31555296"],"confidence":"High","gaps":["Mechanism linking PVM integrity to cyst development not defined","Relative contributions of GRA7 vs. co-deleted GRAs not fully separated"],"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud; RH"}]},{"year":2020,"claim":"Reporter and deletion analyses positioned GRA7 alongside GRA14/GRA15 as a contributor to host NF-κB/RelA activation that restrains parasite expansion.","evidence":"NF-κB luciferase reporter, Δgra7/Δgra14/Δgra15 deletions, RelA translocation, RNA-seq, cytokine and in vivo virulence assays in Pru","pmids":["32849602"],"confidence":"High","gaps":["Direct molecular partner of GRA7 driving RelA translocation not identified","Relationship to the TRAF6/MyD88 axis not reconciled"],"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud"}]},{"year":2020,"claim":"Time-course imaging showed GRA7 localizing to the cyst periphery from early differentiation and persisting in the mature cyst membrane, supporting PVM-to-cyst-membrane continuity.","evidence":"Live imaging and immunofluorescence during in vitro differentiation, saponin permeabilization, and genetic deletion with cyst-wall accumulation readout in Pru","pmids":["31941814"],"confidence":"High","gaps":["Molecular contribution of GRA7 to cyst-wall protein deposition not mechanistically defined","Differential saponin extraction of GRA5 vs. GRA7 unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud"}]},{"year":2020,"claim":"Characterization of GRA60 clarified that GRA7 is one of several parallel effectors (GRA7, GRA60, ROP5/ROP17/ROP18) resisting IRG-mediated PVM disruption.","evidence":"GRA60 deletion, IRG recruitment assay, GRA60-ROP18 co-IP, and in vivo virulence in RH","pmids":["33040458"],"confidence":"Medium","gaps":["How the multiple IRG-resistance effectors are coordinated at the PVM not resolved","GRA7's role here is corroborative rather than primary"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2025,"claim":"Detection of GRA7 in neuron-derived extracellular vesicles and in astrocyte nuclei suggested a route for GRA7 dissemination and host-cell reprogramming beyond the infected cell.","evidence":"LC-MS/MS EV proteomics, nanoparticle tracking, electron microscopy, immunofluorescence of GRA7 in astrocyte nuclei, and astrocyte RNA-seq","pmids":["40523037"],"confidence":"Medium","gaps":["Not yet independently replicated","Mechanism of GRA7 nuclear entry and direct causal role in astrocyte gene changes not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The structural basis of GRA7's PVM membrane insertion and its direct engagement of Irga6, and how a single protein integrates tubulogenesis, IRG neutralization, NF-κB activation, and cyst maturation, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of GRA7 or its complexes","Mechanistic coupling between GRA7's distinct functions unknown","How host phosphorylation states switch GRA7 between roles not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Oligomeric dense granule complex with GRA2, GRA3, GRA5, GRA6 (GRA7 partner)","PVM virulence complex with ROP5, ROP18, ROP2/ROP4 (pseudokinase ROP8/ROP2)","GRA7–Irga6 (GTP-loaded dimer) complex"],"partners":["GRA2","GRA3","GRA5","GRA6","GRA1","ROP2","ROP4","ROP5","ROP18","IRGA6 (HOST)","TRAF6 (HOST)","MYD88 (HOST)","ASC (HOST)","PLD1 (HOST)","PKCΑ (HOST)"],"other_free_text":["Integral membrane protein with hydrophobic anchor at residues 181–202; N-terminal signal peptide and C-terminal transmembrane domain [#1, #3]","Host-cell-dependent phosphorylation; PKCα phosphorylates Ser52 (GRA7-I) and Ser135 (GRA7-III) to control ASC and PLD1 interactions [#6, #10]","C-terminal domain (GRA7-V) mediates TRAF6 binding and ubiquitination [#9]","Uniquely expressed in all life-cycle stages including enteric/coccidian forms, unlike GRA1–6 [#4, #14]","Required for chronic cyst wall maturation and persists in the cyst membrane [#11, #13]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"16630815","id":"PMC_16630815","title":"Toxoplasma gondii sequesters lysosomes from mammalian hosts in the 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and tears in Brazil.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38319921","citation_count":5,"is_preprint":false},{"pmid":"38720960","id":"PMC_38720960","title":"A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.","date":"2024","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/38720960","citation_count":3,"is_preprint":false},{"pmid":"38554777","id":"PMC_38554777","title":"Genetic characterization of Toxoplasma gondii strains isolated from humans living in İzmir, Türkiye.","date":"2024","source":"Indian journal of medical microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/38554777","citation_count":3,"is_preprint":false},{"pmid":"38898997","id":"PMC_38898997","title":"Comparison of the performance of SAG2, GRA6, and GRA7 for serological diagnosis of Toxoplasma gondii infection in 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microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/37502604","citation_count":9,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":52884,"output_tokens":5886,"usd":0.123471,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12903,"output_tokens":6620,"usd":0.115007,"stage2_stop_reason":"end_turn"},"total_usd":0.238478,"stage1_batch_id":"msgbatch_018xTxyBPcQFK9jdgubtxgsj","stage2_batch_id":"msgbatch_01UiG5BTaJP7BvKKL6RLeyv2","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"GRA7 acts as a tubulogenic protein that decorates microtubule-based invaginations of the parasitophorous vacuole membrane (PVM), functioning like a garrote to sequester host endo-lysosomes within the vacuolar space. The parasite actively recruits host microtubules to the PV, and these tubular conduits decorated by GRA7 mediate unidirectional transport and sequestration of host endocytic organelles.\",\n      \"method\": \"Immunoelectron microscopy, fluorescence microscopy, subcellular fractionation, live imaging of host endo-lysosome recruitment to PV\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct imaging and fractionation demonstrating GRA7 decoration of tubular conduits and endo-lysosome sequestration, published in high-impact journal with multiple orthogonal methods\",\n      \"pmids\": [\"16630815\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRA7 (p29) is a dense granule protein that, after host cell invasion, is secreted into the parasitophorous vacuole (PV) and associates with the PV membrane and intravacuolar network. In bradyzoite-infected cells, GRA7 is present in the host cell cytoplasm and is released into culture supernatant, suggesting excretion from cyst-bearing cells. The protein contains a putative N-terminal signal peptide and a C-terminal hydrophobic transmembrane domain, and is expressed in all three intraspecies subgroups.\",\n      \"method\": \"Immunoelectron microscopy, immunofluorescence, Northern blot, ELISA, protein sequencing from tryptic peptides, genomic DNA amplification\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent papers (PMID 9566518 and 9566517) using multiple orthogonal methods (immunoelectron microscopy, immunofluorescence, biochemistry) establishing dense granule localization and post-invasion secretion\",\n      \"pmids\": [\"9566518\", \"9566517\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Ultrastructural immunolocalization demonstrated that GRA7 (P29) traffics from dense granules via the intravacuolar network to the PVM after tachyzoite invasion, associating with the parasite membrane complex, tubular elements of the intravacuolar network, and the parasitophorous vacuolar membrane.\",\n      \"method\": \"Immunogold electron microscopy on thin sections using monoclonal antibody 5-241-178\",\n      \"journal\": \"The journal of histochemistry and cytochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — ultrastructural immunolocalization with quantitative immunogold, single lab but rigorous method establishing trafficking route\",\n      \"pmids\": [\"9815283\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"GRA7 is exclusively membrane-associated in parasites and infected host cells. Triton X-114 partitioning demonstrates GRA7 is an integral membrane protein, with the hydrophobic stretch from amino acids 181–202 serving as a membrane anchor. GRA7 is detected on the surface of intact host cells (originating intracellularly), and additional slower-migrating modified forms of GRA7 appear in membranous fractions from infected host cells, indicating post-exocytosis modification. GRA7 expression level correlates with T. gondii virulence, with a massive decrease in GRA7 synthesis in an in vitro-attenuated variant. GRA7-specific antibodies are present in cerebrospinal fluid during chronic infection, consistent with a role at the parasite-host interface.\",\n      \"method\": \"Proteome analysis, immunoblot, subcellular fractionation, Triton X-114 partitioning, flow cytometry\",\n      \"journal\": \"Microbes and infection\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods (fractionation, detergent partitioning, flow cytometry) in a single lab establishing integral membrane character and host cell surface localization\",\n      \"pmids\": [\"12048027\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"GRA7 is expressed in all infectious life cycle stages of T. gondii (tachyzoite, bradyzoite, merozoite, sporozoite) and is uniquely the only dense granule protein also expressed in enteric (coccidian) stages in the cat gut. During tachyzoite development, GRA7 is secreted and associates with the parasitophorous vacuole. In bradyzoites within mature cysts, GRA7 staining of the cyst wall is reduced compared to tachyzoites. In enteric forms, GRA7 strongly labels PV-containing early asexual and sexual stages. This stage-specific pattern differentiates GRA7 from GRA1–6.\",\n      \"method\": \"Immunocytochemistry with stage-specific markers (SAG1, BAG1), electron microscopy, in vivo infection model (cat and intermediate host)\",\n      \"journal\": \"Parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo immunocytochemistry with double labeling using established stage markers, single lab\",\n      \"pmids\": [\"10503251\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"GRA proteins, including GRA7, form high-molecular-weight oligomeric complexes within the parasite dense granules and within the PV soluble fraction. Affinity purification of HA-FLAG-tagged GRA2 and GRA5 revealed that GRA3, GRA6, and GRA7 are major partners in these complexes. After insertion into PV membranes, GRA2 associates with GRA5 and GRA7, while GRA5 associates with GRA7 only, suggesting progressive loss of partners upon membrane insertion.\",\n      \"method\": \"Affinity chromatography (HA-FLAG tag purification) of knockout parasites complemented with tagged GRA2 or GRA5, conventional chromatography, immunoblot\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — affinity purification from engineered knockout-complemented parasites with multiple GRA partners identified; rigorous genetic background with reciprocal pulls\",\n      \"pmids\": [\"17959262\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GRA7 is phosphorylated in infected host cells but not in extracellular parasites, indicating host cell-dependent phosphorylation. Within 10 min of invasion, GRA7 appears in strand-like structures in the host cytosol that also contain rhoptry proteins. Independently of its phosphorylation state, GRA7 associates with rhoptry proteins ROP2 and ROP4 in infected host cells. GRA7 strands also contain the dense granule proteins GRA1 and GRA3.\",\n      \"method\": \"Immunoprecipitation, immunofluorescence, phosphorylation analysis of extracellular vs. intracellular parasites, co-localization studies\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-immunoprecipitation demonstrating GRA7-ROP2/ROP4 association, phosphorylation established by comparing intra- vs. extracellular conditions, multiple orthogonal methods\",\n      \"pmids\": [\"18809661\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GRA7 exists in a complex with the pseudokinases ROP8/ROP2 and the active kinase ROP18 at the PVM. Individual deletion of gra7 or rop18 partially attenuates virulence in mice, while the double gra7/rop18 mutant is avirulent, placing GRA7 and ROP18 in the same pathway. GRA7 directly binds the active GTP-bound dimer of immunity-related GTPase a6 (Irga6) in a GTP-dependent manner, leading to enhanced Irga6 polymerization, rapid turnover, and eventual disassembly. Increased IRG recruitment and parasite clearance occur in IFN-γ-activated macrophages lacking GRA7 and ROP18.\",\n      \"method\": \"Genetic deletion (single and double knockout), in vivo virulence assays in mice, direct binding assay (GRA7 to Irga6 dimer), IFN-γ-activated macrophage clearance assay, epistasis analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — combination of genetic epistasis (double KO avirulent), direct in vitro binding assay, and functional macrophage clearance assay; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"24390541\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA7 is an additional component of the ROP5/ROP18 kinase complex and associates with the PVM through direct binding to ROP5. Absence of GRA7 results in reduced phosphorylation of Irga6, increased vacuolar IRG protein amounts, and attenuated virulence. GRA7 is strictly an Irga6-specific virulence effector (not targeting other IRG family members), functioning as a regulator for ROP18-specific inactivation of Irga6.\",\n      \"method\": \"Co-immunoprecipitation, genetic deletion analysis, Irga6 phosphorylation assay, in vivo virulence assay, IRG recruitment quantification\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP establishing GRA7-ROP5 interaction, genetic deletion with Irga6 phosphorylation readout, replication of GRA7-IRG axis from PMID 24390541\",\n      \"pmids\": [\"26247512\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA7 induces MyD88-dependent signaling through TRAF6 activation and ROS production, leading to NF-κB-mediated proinflammatory responses in macrophages. GRA7 physically associates with TRAF6 via MyD88. The C-terminal domain of GRA7 (GRA7-V) is sufficient for interaction with and ubiquitination of the RING domain of TRAF6, enabling inflammatory cytokine production. PKCα-independent ROS generation and TRAF6 activation are mutually dependent on GRA7/MyD88 signaling.\",\n      \"method\": \"Co-immunoprecipitation (GRA7-TRAF6 interaction), domain-deletion mapping (GRA7-V), ubiquitination assay, cytokine measurement, MyD88-knockout macrophages\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus domain mapping plus functional ubiquitination assay in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"26553469\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKCα-mediated phosphorylation of GRA7 at Ser52 (GRA7-I domain) regulates GRA7 interaction with the PYD domain of ASC (apoptosis-associated speck-like protein), enabling oligomerization and inflammasome activation for antimicrobial defense against Mycobacterium tuberculosis. GRA7-III domain interacts with the PX domain of phospholipase D1 (PLD1) in a Ser135 phosphorylation-dependent manner (also via PKCα), facilitating PLD1 enzyme activity and phago-lysosomal maturation, contributing to antimicrobial activity.\",\n      \"method\": \"Co-immunoprecipitation (GRA7-ASC, GRA7-PLD1), phosphorylation-site mutagenesis (Ser52, Ser135), PLD1 enzyme activity assay, phago-lysosomal maturation assay, inflammasome activation assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro binding with mutagenesis of phosphorylation sites, enzyme activity assay, and functional phagolysosomal readout; multiple orthogonal methods in one study\",\n      \"pmids\": [\"28125719\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deletion of PVM-associated GRA7 (along with GRA3, GRA8, GRA14) in the type II Prugniaud strain induces severe defects in chronic cyst development in vivo without affecting in vitro growth rate or in vitro differentiation. GRA7 deletion parasites show reduced acute virulence. GRA7 (along with ROP5, ROP18) suppresses MHC-I antigen presentation of PV-localized parasite antigens to CD8+ T cells, with suppression correlated to prevention of PV disruption by IFN-γ-activated host IRG effectors.\",\n      \"method\": \"Genetic deletion in type II Pru strain, in vivo chronic infection model (mouse), in vitro differentiation assay, MHC-I antigen presentation assay, IRG recruitment assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic deletion with in vivo cyst formation, multiple GRA deletions compared, functional antigen presentation assay; replicated across two papers (PMID 31266861 and 31555296)\",\n      \"pmids\": [\"31266861\", \"31555296\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud; RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA7 and GRA14 (but not GRA15 alone) are partially involved in NF-κB activation; all three (GRA7, GRA14, GRA15) contribute to nuclear translocation of RelA. Deletion of GRA7, GRA14, and GRA15 decreases IL-6 levels in macrophages and downregulates NF-κB-mediated gene expression. Luciferase reporter assays confirm GRA7 overexpression activates NF-κB-dependent transcription. Deletion of GRA7 in the type II Pru strain increases parasite virulence, suggesting GRA7-induced host NF-κB immunity limits parasite expansion.\",\n      \"method\": \"Luciferase reporter assay (NF-κB), genetic deletion (Δgra7, Δgra14, Δgra15), RelA nuclear translocation assay, RNA-seq, cytokine measurement, in vivo virulence assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase reporter plus genetic deletion plus RNA-seq plus in vivo virulence; multiple orthogonal methods in one study\",\n      \"pmids\": [\"32849602\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA5 and GRA7 localize to the cyst periphery as early as 6 h after in vitro differentiation and remain at the cyst membrane and cyst wall region throughout cyst maturation (up to day 10), supporting the model that the PVM remains intact and develops into the cyst membrane. In mature cysts, saponin permeabilization selectively removes GRA5 but not GRA7 from the cyst membrane. Deletion of PVM-localized GRAs including GRA7 reduces the rate of cyst wall protein accumulation at the cyst periphery.\",\n      \"method\": \"Live imaging and immunofluorescence of GRA5/GRA7 during in vitro differentiation, saponin permeabilization assay, genetic deletion of individual GRAs with cyst wall protein accumulation readout\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — time-course live imaging plus biochemical permeabilization plus genetic deletion with functional cyst wall readout; multiple methods in one study\",\n      \"pmids\": [\"31941814\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"GRA7 is expressed in tachyzoites, bradyzoites, and coccidian/enteric stages (merozoites), whereas GRA1–6 are absent in merozoites and enteric stages. In tachyzoites, GRA7 extensively labels the PV; in bradyzoites within cysts, the cyst wall shows variable staining. This unique expression in all stages is reflected in the differences in PV structure observed by electron microscopy across life cycle stages.\",\n      \"method\": \"Immunocytochemistry with panel of molecular markers, electron microscopy, in vivo infections in cat and intermediate host\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo immunocytochemistry with multiple markers across life stages, corroborates PMID 10503251\",\n      \"pmids\": [\"15003495\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GRA7 protein is packaged into extracellular vesicles (EVs) released from Toxoplasma-infected neurons, and GRA7 can be observed within intracellular EVs and in the nuclei of GRA7+ EV-containing astrocytes after incubation with infected-neuron EVs. EVs from infected neurons altered astrocyte gene expression toward pro-inflammatory signatures and downregulated GLT-1 glutamate transporter expression.\",\n      \"method\": \"LC-MS/MS proteomic characterization of EVs, immunofluorescence (GRA7 in astrocyte nuclei), nanoparticle tracking analysis, transmission electron microscopy, RNA-seq of astrocytes\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — proteomic identification plus immunofluorescence in astrocytes, single lab, novel finding about GRA7 in EVs and nuclear translocation not yet independently replicated\",\n      \"pmids\": [\"40523037\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA60, a novel dense granule effector at the vacuolar space and PVM, contributes to resistance to IRG recruitment independently of GRA7. GRA60 is found associated with ROP18. Deletion of GRA60 in an RHΔrop5 background leads to IRG recruitment comparable to single knockouts, demonstrating GRA60 and ROP5 act in the same pathway. This study contextualizes GRA7 as one member of a broader virulence complex (GRA7, ROP5, ROP17, ROP18) resisting IRG-mediated PVM disruption.\",\n      \"method\": \"Genetic deletion of GRA60, IRG recruitment assay, co-immunoprecipitation (GRA60-ROP18), in vivo virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GRA7 role confirmed as part of IRG-resistance complex; GRA60 data is primary finding but GRA7's role in the complex is corroborated\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"GRA7 is a dense granule protein expressed across all T. gondii life stages that is secreted after invasion into the parasitophorous vacuole (PV), where it integrates as an integral membrane protein into the PV membrane (PVM), intravacuolar network tubules, and cyst membrane; it acts as a tubulogenic garrote to sequester host endo-lysosomes within the vacuolar space, forms oligomeric complexes with GRA2, GRA3, GRA5, and GRA6, associates with rhoptry proteins ROP2, ROP4, ROP5, and ROP18 in a virulence complex that directly binds and disassembles the GTP-loaded Irga6 dimer to protect the PVM from IRG-mediated destruction and suppress MHC-I antigen presentation, activates host NF-κB and TRAF6/MyD88 innate immune signaling through its C-terminal domain, undergoes PKCα-mediated phosphorylation that controls interactions with ASC (inflammasome) and phospholipase D1 (phago-lysosomal maturation), and is required for normal cyst wall maturation during chronic infection.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA7 is a Toxoplasma gondii dense granule protein secreted after host cell invasion into the parasitophorous vacuole (PV), where it traffics via the intravacuolar network to the PV membrane (PVM) and integrates as an integral membrane protein through a hydrophobic membrane anchor (residues 181–202) [#1, #2, #3]. It is expressed across all infectious life stages, uniquely including the enteric coccidian stages that lack other dense granule proteins, and persists at the cyst periphery during chronic infection [#4, #14, #13]. At the PVM, GRA7 decorates microtubule-based tubular invaginations and acts as a tubulogenic \\\"garrote\\\" that sequesters host endo-lysosomes within the vacuolar space [#0], and it assembles into high-molecular-weight oligomeric complexes with other GRA proteins including GRA2, GRA3, GRA5, and GRA6 [#5]. GRA7 is a central effector of a virulence complex with the rhoptry proteins ROP2/ROP4, ROP5, and the active kinase ROP18: it associates with the PVM through direct binding to ROP5 and directly binds the GTP-loaded Irga6 dimer in a GTP-dependent manner, driving Irga6 phosphorylation, polymerization, and disassembly to protect the PVM from IRG-mediated destruction [#6, #7, #8]. Genetic epistasis places GRA7 and ROP18 in the same pathway, and loss of GRA7 (with ROP5/ROP18) permits IRG recruitment, PV disruption, and increased MHC-I presentation of parasite antigens to CD8+ T cells [#7, #11]. GRA7 also engages host innate immune signaling, activating MyD88/TRAF6-dependent NF-κB proinflammatory responses through its C-terminal domain [#9, #12], and undergoes PKCα-mediated phosphorylation (Ser52, Ser135) that governs its interactions with ASC and phospholipase D1 in antimicrobial defense [#10]. GRA7 is required for normal chronic cyst wall maturation in vivo [#11, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing GRA7 as a secreted dense granule protein answered where it acts, defining its localization at the PV membrane and intravacuolar network after invasion.\",\n      \"evidence\": \"Immunoelectron microscopy, immunofluorescence, and biochemistry on tachyzoite- and bradyzoite-infected cells, plus immunogold trafficking analysis\",\n      \"pmids\": [\"9566518\", \"9566517\", \"9815283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane topology and mode of PVM insertion not yet resolved\", \"Functional consequence of secretion not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Documenting GRA7 expression across all life-cycle and enteric stages distinguished it from GRA1–6 and implicated it in every infectious form.\",\n      \"evidence\": \"Immunocytochemistry with stage-specific markers and electron microscopy in cat and intermediate-host infections\",\n      \"pmids\": [\"10503251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stage-specific function not defined\", \"Reduced cyst-wall staining in bradyzoites left unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Biochemical characterization resolved GRA7 as an integral membrane protein with a defined anchor and linked its expression to parasite virulence.\",\n      \"evidence\": \"Triton X-114 partitioning, subcellular fractionation, and flow cytometry, plus correlation with an attenuated variant\",\n      \"pmids\": [\"12048027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of post-exocytosis modifications not identified\", \"Mechanistic basis of the virulence correlation not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Confirmation of GRA7 expression in tachyzoites, bradyzoites, and merozoites reinforced its unique pan-stage distribution relative to other GRAs.\",\n      \"evidence\": \"Immunocytochemistry with marker panel, electron microscopy, and in vivo infections\",\n      \"pmids\": [\"15003495\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not test function in any stage\", \"Cyst-wall staining variability unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery of GRA7-decorated microtubule tubules answered how the PV manipulates host endocytic organelles, defining a tubulogenic sequestration role.\",\n      \"evidence\": \"Immunoelectron and fluorescence microscopy, fractionation, and live imaging of host endo-lysosome recruitment to the PV\",\n      \"pmids\": [\"16630815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of tubule formation by GRA7 not defined\", \"Contribution of sequestration to nutrient acquisition not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Affinity purification defined GRA7 as a member of oligomeric GRA complexes, showing how dense granule proteins assemble before and after membrane insertion.\",\n      \"evidence\": \"HA-FLAG affinity purification from knockout-complemented parasites with reciprocal pulls and immunoblot\",\n      \"pmids\": [\"17959262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural arrangement of complexes unknown\", \"Functional role of GRA7 within these complexes not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Detection of host-dependent phosphorylation and rhoptry-protein association linked GRA7 to ROP2/ROP4 in host-cytosolic strands, foreshadowing its virulence-complex role.\",\n      \"evidence\": \"Immunoprecipitation, immunofluorescence colocalization, and comparison of extracellular vs. intracellular parasite phosphorylation\",\n      \"pmids\": [\"18809661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for phosphorylation not identified here\", \"Functional significance of GRA7-ROP2/ROP4 strands not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Genetic epistasis and direct binding assays revealed GRA7's core effector mechanism: disabling the host Irga6 GTPase to protect the PVM.\",\n      \"evidence\": \"Single and double gra7/rop18 knockouts, in vivo virulence assays, direct GRA7–Irga6 dimer binding assay, and IFN-γ macrophage clearance in RH strain\",\n      \"pmids\": [\"24390541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of GRA7–Irga6 recognition not resolved\", \"Whether GRA7 targets IRGs beyond Irga6 not addressed here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defining GRA7 as a ROP5-binding component of the ROP5/ROP18 kinase complex answered how GRA7 is recruited to the PVM and regulates Irga6-specific inactivation.\",\n      \"evidence\": \"Reciprocal co-IP, genetic deletion, Irga6 phosphorylation and IRG recruitment quantification, and in vivo virulence\",\n      \"pmids\": [\"26247512\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why GRA7 is Irga6-specific and not broader across IRGs not explained mechanistically\", \"Strain background of this study unspecified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapping the GRA7 C-terminal domain to TRAF6 defined a distinct role activating host MyD88/NF-κB proinflammatory signaling.\",\n      \"evidence\": \"Co-IP, GRA7-V domain-deletion mapping, TRAF6 ubiquitination assay, and cytokine measurement in MyD88-knockout macrophages\",\n      \"pmids\": [\"26553469\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PVM-integral GRA7 accesses cytosolic TRAF6 not resolved\", \"In vivo relevance of the TRAF6 axis not established here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of PKCα phosphosites (Ser52, Ser135) governing GRA7 binding to ASC and PLD1 connected GRA7 phosphorylation to inflammasome and phago-lysosomal antimicrobial defense.\",\n      \"evidence\": \"Co-IP, phosphosite mutagenesis, PLD1 enzyme activity, phago-lysosomal maturation, and inflammasome activation assays\",\n      \"pmids\": [\"28125719\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether these host-defense functions operate during Toxoplasma infection itself vs. heterologous contexts not delineated\", \"Physiological trigger for PKCα phosphorylation not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Deletion studies in the type II Pru strain established GRA7's requirement for chronic cyst development and its role in suppressing MHC-I antigen presentation.\",\n      \"evidence\": \"Genetic deletion of PVM-associated GRAs, in vivo chronic infection, MHC-I presentation and IRG recruitment assays in Pru and RH\",\n      \"pmids\": [\"31266861\", \"31555296\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking PVM integrity to cyst development not defined\", \"Relative contributions of GRA7 vs. co-deleted GRAs not fully separated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud; RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Reporter and deletion analyses positioned GRA7 alongside GRA14/GRA15 as a contributor to host NF-κB/RelA activation that restrains parasite expansion.\",\n      \"evidence\": \"NF-κB luciferase reporter, Δgra7/Δgra14/Δgra15 deletions, RelA translocation, RNA-seq, cytokine and in vivo virulence assays in Pru\",\n      \"pmids\": [\"32849602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular partner of GRA7 driving RelA translocation not identified\", \"Relationship to the TRAF6/MyD88 axis not reconciled\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Time-course imaging showed GRA7 localizing to the cyst periphery from early differentiation and persisting in the mature cyst membrane, supporting PVM-to-cyst-membrane continuity.\",\n      \"evidence\": \"Live imaging and immunofluorescence during in vitro differentiation, saponin permeabilization, and genetic deletion with cyst-wall accumulation readout in Pru\",\n      \"pmids\": [\"31941814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular contribution of GRA7 to cyst-wall protein deposition not mechanistically defined\", \"Differential saponin extraction of GRA5 vs. GRA7 unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Characterization of GRA60 clarified that GRA7 is one of several parallel effectors (GRA7, GRA60, ROP5/ROP17/ROP18) resisting IRG-mediated PVM disruption.\",\n      \"evidence\": \"GRA60 deletion, IRG recruitment assay, GRA60-ROP18 co-IP, and in vivo virulence in RH\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How the multiple IRG-resistance effectors are coordinated at the PVM not resolved\", \"GRA7's role here is corroborative rather than primary\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Detection of GRA7 in neuron-derived extracellular vesicles and in astrocyte nuclei suggested a route for GRA7 dissemination and host-cell reprogramming beyond the infected cell.\",\n      \"evidence\": \"LC-MS/MS EV proteomics, nanoparticle tracking, electron microscopy, immunofluorescence of GRA7 in astrocyte nuclei, and astrocyte RNA-seq\",\n      \"pmids\": [\"40523037\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not yet independently replicated\", \"Mechanism of GRA7 nuclear entry and direct causal role in astrocyte gene changes not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of GRA7's PVM membrane insertion and its direct engagement of Irga6, and how a single protein integrates tubulogenesis, IRG neutralization, NF-κB activation, and cyst maturation, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of GRA7 or its complexes\", \"Mechanistic coupling between GRA7's distinct functions unknown\", \"How host phosphorylation states switch GRA7 between roles not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140272\", \"supporting_discovery_ids\": [7, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005515\", \"supporting_discovery_ids\": [5, 6, 7, 8, 9, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051607\", \"supporting_discovery_ids\": [7, 8, 11, 16], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [1, 2, 3, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016020\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}]},\n      {\"term_id\": \"GO:0020007\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"BK\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:1903561\", \"supporting_discovery_ids\": [15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051873\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0043123\", \"supporting_discovery_ids\": [9, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0002376\", \"supporting_discovery_ids\": [7, 8, 9, 10, 11, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Oligomeric dense granule complex with GRA2, GRA3, GRA5, GRA6 (GRA7 partner)\",\n      \"PVM virulence complex with ROP5, ROP18, ROP2/ROP4 (pseudokinase ROP8/ROP2)\",\n      \"GRA7–Irga6 (GTP-loaded dimer) complex\"\n    ],\n    \"partners\": [\n      \"GRA2\", \"GRA3\", \"GRA5\", \"GRA6\", \"GRA1\", \"ROP2\", \"ROP4\", \"ROP5\", \"ROP18\",\n      \"Irga6 (host)\", \"TRAF6 (host)\", \"MyD88 (host)\", \"ASC (host)\", \"PLD1 (host)\", \"PKCα (host)\"\n    ],\n    \"other_free_text\": [\n      \"Integral membrane protein with hydrophobic anchor at residues 181–202; N-terminal signal peptide and C-terminal transmembrane domain [#1, #3]\",\n      \"Host-cell-dependent phosphorylation; PKCα phosphorylates Ser52 (GRA7-I) and Ser135 (GRA7-III) to control ASC and PLD1 interactions [#6, #10]\",\n      \"C-terminal domain (GRA7-V) mediates TRAF6 binding and ubiquitination [#9]\",\n      \"Uniquely expressed in all life-cycle stages including enteric/coccidian forms, unlike GRA1–6 [#4, #14]\",\n      \"Required for chronic cyst wall maturation and persists in the cyst membrane [#11, #13]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_203310","name":"GRA7","product":"dense granule protein GRA7","uniprot_acc":"A0A125YI90","chromosome":"TGME49_chrVIIa","start_pos":2604221},{"strain":"GT1","gene_id":"TGGT1_203310","name":null,"product":"dense granule protein GRA7","uniprot_acc":null,"chromosome":"TGGT1_chrVIIa","start_pos":2502941},{"strain":"RH88","gene_id":"TGRH88_035710","name":null,"product":"dense granule protein GRA7","uniprot_acc":null,"chromosome":"CM023088","start_pos":2697725},{"strain":"VEG","gene_id":"TGVEG_203310","name":null,"product":"dense granule protein GRA7","uniprot_acc":"A0A0F7UZ13","chromosome":"TGVEG_chrVIIa","start_pos":2692320}]}