{"gene":"TG_275440","gene_symbol":"GRA6","run_date":"2026-09-08T16:59:57+00:00","timeline":{"discoveries":[{"year":1995,"finding":"GRA6 is a 32 kDa dense granule antigen that is localized to the dense granules of tachyzoites and, following secretion, associates with the intravacuolar network of the parasitophorous vacuole. The gene encodes a 230-amino-acid polypeptide with two hydrophobic transmembrane-like domains; the N-terminal domain lacks classical signal peptide features, and the C-terminal hydrophilic region is glycine-rich (24%), suggesting a structural role in the network.","method":"cDNA cloning, gene sequencing, immunolocalization with specific antiserum (immunofluorescence and immunoelectron microscopy), primer extension analysis","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct subcellular localization by immunoelectron microscopy combined with gene/protein characterization; foundational characterization paper replicated by subsequent studies","pmids":["7637717"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"Following secretion into the parasitophorous vacuole, GRA6 is rapidly translocated to the posterior end of the parasite where it binds to a cluster of multi-lamellar vesicles that give rise to the intravacuolar network. GRA6 association with network membranes is predominantly mediated by hydrophobic interactions, and a phosphorylated form of GRA6 shows increased membrane association. Cross-linking studies established that GRA4 and GRA6 specifically interact with GRA2 to form a multimeric complex stably associated with the intravacuolar network.","method":"Cell fractionation, denaturing agent treatment, cross-linking studies, immunofluorescence microscopy, immunoelectron microscopy","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical methods (fractionation, cross-linking, denaturing agents) in a single rigorous study establishing both localization and protein-protein interactions","pmids":["10498186"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"GRA6 is required for the normal nanotubular conformation of the intravacuolar network (IVN). Deletion of GRA6 (Δgra6 mutant) leads to an altered mature network characterized by small vesicles instead of elongated nanotubules; however, the initial formation of the posterior organizing center was normal in Δgra6 parasites (in contrast to Δgra2 parasites). GRA6 also failed to localize properly to the posterior organizing center in Δgra2 cells, indicating GRA2-dependent localization of GRA6.","method":"Gene knockout (Δgra6 and Δgra2 mutants), immunofluorescence microscopy, electron microscopy, complementation with mutated GRA2 forms","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — specific gene knockout with defined ultrastructural phenotype, epistasis analysis with Δgra2, complementation experiments with mutagenized constructs","pmids":["12134078"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"GRA6 is the source of the immunodominant and protective decapeptide HF10 presented by the H-2L(d) MHC class I molecule in BALB/c mice. Presentation of the HF10-H-2L(d) ligand required proteolysis by ERAAP (the endoplasmic reticulum aminopeptidase associated with antigen processing), and expansion of protective CD8+ T cell populations was impaired in ERAAP-deficient mice infected with T. gondii.","method":"Peptide identification from infected cells, T cell assays, ERAAP knockout mouse infection, CD8+ T cell expansion assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct identification of the epitope source, genetic knockout of processing enzyme (ERAAP) with defined immunological phenotype, in vivo protection assay","pmids":["18587399"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"GRA6 (together with GRA4) plays a functional role in brain cyst development and latent infection in type II T. gondii. Deletion of GRA6 in type II Δku80 parasites drastically reduced brain tissue cyst burdens in mice. Complementation with a functional GRA6 allele at the endogenous UPRT locus significantly restored cyst burdens, confirming the specific role of GRA6.","method":"Gene knockout in type II Δku80 background, mouse infection model with brain cyst counting, genetic complementation","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — specific gene deletion with defined in vivo phenotype, genetic complementation restoring the phenotype, rigorous controls","pmids":["21531875"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Pru (type II)"}]},{"year":2013,"finding":"The location of the GRA6 peptide epitope at the C-terminus of the GRA6 antigenic precursor determines immunodominance and optimal MHC I processing. Parasite burden is controlled by GRA6-specific CD8 T cells but not by responses to subdominant GRA4- and ROP7-derived epitopes. Optimal processing and immunodominance were determined by the C-terminal position of the epitope, not by peptide affinity for MHC I or precursor T cell frequency.","method":"Genetically modified parasites expressing epitope-repositioned GRA6 variants, in vivo CD8 T cell response assays, parasite burden measurements","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetically modified parasites with epitope repositioning, multiple in vivo readouts, direct functional comparison of dominant vs subdominant responses","pmids":["23818852"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"The polymorphic dense granule protein GRA6 selectively activates the host transcription factor NFAT4 via CAMLG. GRA6 overexpression robustly and selectively activated NFAT4. Infection with wild-type but not GRA6-deficient parasites induced NFAT4 activation. GRA6-deficient parasites exhibited reduced virulence in local infection. NFAT4-deficient mice showed prolonged survival, decreased CD11b+Ly6G+ cell recruitment, and impaired expression of chemokines Cxcl2 and Ccl2. Type I parasites caused significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C-terminus of GRA6.","method":"GRA6 overexpression, GRA6 gene knockout, NFAT4 knockout mice, in vivo infection assays, flow cytometry, chemokine expression analysis, NFAT inhibitor treatment","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic tools (overexpression, knockout parasite, host knockout), in vivo functional readouts, identification of CAMLG as the molecular intermediary, strain-specific polymorphism linked to activity","pmids":["25225460"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"GRA6 membrane association in the parasitophorous vacuole regulates its accessibility to the MHC class I antigen presentation pathway. Using a cell-free minimal system, GRA6 and GRA2 were shown to shape membrane tubules. Insertion of GRA6 in the PV membrane is key for immunogenicity, whereas association of GRA6 with the IVN limits MHC I presentation and curtails GRA6-specific CD8 T cell responses in mice.","method":"Cell-free minimal membrane system reconstitution, MHC I presentation assays in infected cells, CD8 T cell response quantification in mice, GRA6/GRA2 knockout parasites","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free reconstitution of membrane tubule formation, combined with in cellulo antigen presentation assays and in vivo T cell responses, multiple orthogonal methods","pmids":["26628378"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"GRA6 (together with GRA2) contributes to host vesicle sequestration within the parasitophorous vacuole. Rab11A vesicles concentrate at the intravacuolar network (IVN), but this concentration is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that IVN tubules stabilized by GRA2 and GRA6 contribute to host vesicle sequestration.","method":"Genetic knockout (Δgra2, Δgra2Δgra6), correlative light and electron microscopy, live imaging of GFP-Rab11A vesicles","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double-knockout with defined vesicle sequestration phenotype, but the Δgra6 single knockout effect on Rab11A was not directly tested alone; conclusion on GRA6 is partly inferred from double-KO","pmids":["29070609"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1996,"finding":"The promoter of GRA6 contains cis-acting elements critical for expression. Deletion analysis mapped optimal GRA6 promoter activity to a region -146 to -27 bp upstream of the transcription start site. Repeated motifs (A/TGAGACG) found in either orientation within these proximal regions are critical elements for promoter activity of GRA6 (and other GRA genes).","method":"Transient transformation with CAT reporter gene constructs, deletion analysis of promoter regions, mutation analysis of cis-elements","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter gene assays with deletion and mutation analysis in a single lab, defines promoter elements functionally","pmids":["8858595"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"finding":"GRA6 sequence polymorphisms correlate with T. gondii strain type and murine virulence. Sequence alignment of the GRA6 coding region (690 bp) across 30 strains from 8 zymodemes identified 24 polymorphic nucleotide positions distinguishing types I, II, and III. Type II strains have unique deletions of 15 bp and 3 bp causing loss of 6 amino acids. A PCR-RFLP method using MseI was developed to distinguish the three lineages based on GRA6 polymorphisms.","method":"PCR amplification and DNA sequencing of GRA6 coding region from 30 strains, sequence alignment, PCR-RFLP with MseI enzyme","journal":"International journal for parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — sequence-based characterization across multiple strains establishing genotype-phenotype correlation, but mechanism is correlational rather than experimentally dissected","pmids":["10779578"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1997,"finding":"In sporozoites, GRA6 protein is present in dense granules and is detected in oocyst extracts, but unlike tachyzoite behavior, GRA6 (along with GRA1, GRA2, GRA4) is retained within cytoplasmic granules during sporozoite invasion of host cells and is not secreted into the first parasitophorous vacuole (PV1). This establishes a stage-specific difference in GRA6 secretion behavior.","method":"Immunofluorescence with anti-GRA antibodies on sporozoite-infected cells, comparison of tachyzoite vs sporozoite secretion, oocyst extract immunoblot","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — immunolocalization studies comparing two life stages, single lab but with multiple antibodies and consistent results","pmids":["9353039"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"finding":"The N-terminal hydrophilic region of GRA6 (but not the C-terminal region) is recognized by human anti-Toxoplasma sera. Recombinant GST-GRA6-Nt ELISA achieved 96% sensitivity for detection of T. gondii IgG in human sera, demonstrating that the N-terminal hydrophilic domain is the immunodominant region of GRA6 in humans.","method":"Recombinant protein expression in E. coli (separate N- and C-terminal domains), immunoblot with pooled human sera, ELISA with 100 positive and 98 negative human sera","journal":"Clinical and diagnostic laboratory immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain-mapping by differential recombinant protein reactivity with a well-characterized serum panel, single lab","pmids":["10882660"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"GRA12 behaves similarly to GRA6 in that it is secreted into the PV from the anterior pole, transits to the posterior invagination where the IVN assembles, and finally resides in the vacuolar space associated with the mature nanotubular network. GRA12 fails to localize at the parasite posterior end in the absence of GRA2, similar to GRA6. Affinity chromatography using tagged GRA2 co-purified GRA12 with GRA2 in both parasite and PV soluble fractions, placing GRA12 in the same GRA protein complex that includes GRA6.","method":"Immunofluorescence, immunoelectron microscopy, GRA2 knockout analysis, affinity chromatography with tagged GRA2","journal":"International journal for parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — GRA12 characterization paper that provides additional evidence for GRA6 complex membership via affinity chromatography; GRA6 is a comparator, not the primary subject","pmids":["18840447"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"GRA6 is a dense granule protein secreted into the Toxoplasma gondii parasitophorous vacuole where it associates with the intravacuolar network (IVN) via hydrophobic interactions and a GRA2-dependent posterior organizing center; it stabilizes IVN nanotubular architecture, facilitates host Rab vesicle sequestration, forms a multimeric complex with GRA2 and GRA4, promotes brain cyst development during chronic infection, and acts as a host-signaling effector by activating NFAT4 through CAMLG in a strain-polymorphism-dependent manner, while also serving as a source of the immunodominant CD8+ T cell epitope HF10 whose access to MHC I presentation is regulated by GRA6 membrane topology within the PV."},"narrative":{"mechanistic_narrative":"GRA6 is a dense granule protein of Toxoplasma gondii that is secreted into the parasitophorous vacuole (PV), where it functions both as a structural organizer of the intravacuolar network (IVN) and as a strain-polymorphic effector of host signaling and antigen presentation [PMID:7637717, PMID:25225460, PMID:26628378]. After secretion from the anterior pole, GRA6 is translocated to the posterior end of the parasite and associates with the nascent IVN membranes predominantly through hydrophobic interactions, forming a stable multimeric complex with GRA2 and GRA4 [PMID:10498186]. Its correct posterior localization depends on GRA2, and GRA6 is in turn required for the mature nanotubular conformation of the IVN, since Δgra6 vacuoles accumulate small vesicles rather than elongated tubules [PMID:12134078]; in a reconstituted cell-free system GRA6 and GRA2 are sufficient to shape membrane tubules [PMID:26628378]. This GRA2/GRA6-stabilized network contributes to sequestration of host Rab11A vesicles within the PV [PMID:29070609] and promotes brain tissue cyst development during chronic type II infection [PMID:21531875]. GRA6 also acts as a host-manipulating effector, selectively activating the transcription factor NFAT4 through CAMLG in a manner dependent on a C-terminal polymorphism that makes type I strains far more potent than type II, driving myeloid cell recruitment and chemokine induction [PMID:25225460]. Independently, GRA6 supplies the immunodominant, ERAAP-dependent decapeptide HF10 presented by H-2Ld to protective CD8+ T cells, with immunodominance dictated by the C-terminal position of the epitope [PMID:18587399, PMID:23818852], and epitope accessibility governed by GRA6 membrane topology—PV-membrane insertion favors presentation whereas IVN association limits it [PMID:26628378].","teleology":[{"year":1995,"claim":"Establishing that GRA6 is a dense granule antigen that relocates to the intravacuolar network defined it as a secreted structural component of the PV rather than a purely cytosolic parasite protein.","evidence":"cDNA cloning/sequencing plus immunofluorescence and immunoelectron microscopy localization","pmids":["7637717"],"confidence":"High","gaps":["Function of the two hydrophobic domains and glycine-rich C-terminus not tested","No interacting partners identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1996,"claim":"Mapping cis-acting promoter elements answered how GRA6 expression is transcriptionally controlled, identifying repeated A/TGAGACG motifs shared with other GRA genes.","evidence":"CAT reporter deletion and mutation analysis in transient transformation","pmids":["8858595"],"confidence":"Medium","gaps":["Trans-acting factors binding these motifs not identified","Regulation across life stages not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1997,"claim":"Comparing life stages revealed that GRA6 secretion behavior is stage-specific, being retained in cytoplasmic granules during sporozoite invasion rather than secreted into the vacuole.","evidence":"Immunofluorescence and immunoblot comparing sporozoite versus tachyzoite behavior","pmids":["9353039"],"confidence":"Medium","gaps":["Molecular basis of stage-specific retention unknown","Consequence for sporozoite PV biology untested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"Biochemical dissection of GRA6 membrane association showed it binds IVN membranes via hydrophobic interactions and forms a defined complex with GRA2 and GRA4, moving from localization to a molecular assembly model.","evidence":"Cell fractionation, denaturing treatment, cross-linking, and electron microscopy","pmids":["10498186"],"confidence":"High","gaps":["Stoichiometry and interface of the GRA2/GRA4/GRA6 complex not resolved","Role of GRA6 phosphorylation not functionally defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Sequencing GRA6 across strains established a genotype-phenotype correlation, linking GRA6 polymorphisms to strain lineage and murine virulence and providing a genotyping tool.","evidence":"PCR/sequencing of GRA6 across 30 strains and PCR-RFLP with MseI","pmids":["10779578"],"confidence":"Medium","gaps":["Correlation is not a demonstrated causal mechanism","Functional consequence of specific polymorphisms not tested here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Domain mapping of the human antibody response identified the N-terminal hydrophilic region as the immunodominant B-cell region of GRA6, informing serodiagnostic use.","evidence":"Reactivity of recombinant N- versus C-terminal domains with human sera by immunoblot and ELISA","pmids":["10882660"],"confidence":"Medium","gaps":["Does not address T-cell epitope location","Single serum panel"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"Knockout epistasis established that GRA6 is required for the mature nanotubular architecture of the IVN and that its posterior localization is GRA2-dependent, defining a hierarchy within the network-building machinery.","evidence":"Δgra6 and Δgra2 knockouts, EM/IF ultrastructural analysis, complementation with mutant GRA2","pmids":["12134078"],"confidence":"High","gaps":["Mechanism by which GRA6 remodels vesicles into tubules not resolved at this stage","Consequence of altered IVN for parasite fitness not measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Identifying GRA6 as the source of the ERAAP-processed HF10/H-2Ld epitope explained a major immunodominant CD8+ T cell response and linked antigen processing machinery to protective immunity.","evidence":"Epitope identification, T cell assays, and ERAAP-knockout mouse infection","pmids":["18587399"],"confidence":"High","gaps":["Why GRA6-derived HF10 is preferentially processed not yet explained","Restricted to BALB/c H-2Ld context"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Characterization of GRA12 showed it co-purifies with GRA2 and shares GRA6's trafficking route, extending the composition of the GRA2-anchored IVN complex that includes GRA6.","evidence":"IF/immunoEM, GRA2-knockout analysis, and affinity chromatography with tagged GRA2","pmids":["18840447"],"confidence":"Medium","gaps":["Direct GRA6-GRA12 interaction not demonstrated","GRA6 is a comparator rather than the primary subject"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Deletion and complementation in type II parasites established that GRA6 promotes brain cyst development, linking an IVN structural protein to chronic latent infection.","evidence":"Δgra6 in type II Δku80, mouse brain cyst counting, and complementation at the UPRT locus","pmids":["21531875"],"confidence":"High","gaps":["Whether the cyst defect reflects IVN disruption or another GRA6 activity is unresolved","Molecular pathway to cyst formation not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"Pru (type II)"}]},{"year":2013,"claim":"Epitope-repositioning experiments showed that GRA6 immunodominance is dictated by the C-terminal position of HF10, not by MHC affinity or precursor frequency, defining a positional rule for antigen presentation and protection.","evidence":"Epitope-repositioned GRA6 variant parasites, in vivo CD8 T cell responses, and parasite burden measurement","pmids":["23818852"],"confidence":"High","gaps":["Mechanistic basis of the C-terminal positional advantage not fully defined","Generality to other GRA epitopes untested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Discovery that GRA6 selectively activates NFAT4 via CAMLG in a strain-polymorphism-dependent manner recast GRA6 as a host-signaling effector controlling immune cell recruitment and virulence.","evidence":"GRA6 overexpression and knockout, NFAT4-knockout mice, chemokine analysis, and NFAT inhibitor treatment","pmids":["25225460"],"confidence":"High","gaps":["How PV-resident GRA6 engages host CAMLG topologically not resolved","Precise polymorphic residues driving type I vs type II potency not enumerated here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Cell-free reconstitution and presentation assays unified GRA6 structural and immunological roles, showing GRA6/GRA2 shape membrane tubules and that GRA6 membrane topology (PV membrane insertion vs IVN association) gates its MHC I accessibility.","evidence":"Cell-free minimal membrane system, MHC I presentation assays, in vivo CD8 T cell responses, GRA6/GRA2 knockouts","pmids":["26628378"],"confidence":"High","gaps":["What controls partitioning of GRA6 between PV membrane and IVN in vivo is unresolved","Link between topology and NFAT4 signaling not directly tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Double-knockout imaging tied the GRA2/GRA6-stabilized IVN to host vesicle sequestration, connecting network architecture to nutrient/host-vesicle acquisition.","evidence":"Δgra2 and Δgra2Δgra6 parasites, correlative light-electron microscopy, and live GFP-Rab11A imaging","pmids":["29070609"],"confidence":"Medium","gaps":["Δgra6 single-knockout effect on Rab11A not tested alone","Functional consequence of Rab11A sequestration for the parasite not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How GRA6 is partitioned between the PV membrane and the IVN in vivo, and how this topology mechanistically couples its structural, host-signaling (NFAT4/CAMLG), and antigen-presentation functions, remains unresolved.","evidence":"No direct experimental resolution in the available corpus","pmids":[],"confidence":"Low","gaps":["Regulators of GRA6 membrane topology unknown","Structural interface of the GRA2/GRA4/GRA6 complex undetermined","Direct GRA6-CAMLG interaction and its topological requirement not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["GRA2-GRA4-GRA6 multimeric intravacuolar network complex","GRA2-associated IVN complex including GRA12"],"partners":["GRA2","GRA4","GRA12 (CO-COMPLEX VIA GRA2)","CAMLG (HOST)","NFAT4 (HOST, ACTIVATED DOWNSTREAM)","ERAAP (HOST ANTIGEN-PROCESSING AMINOPEPTIDASE)"],"other_free_text":["230-aa polypeptide with two hydrophobic transmembrane-like domains and a glycine-rich C-terminal hydrophilic region [#0]","N-terminal hydrophilic region is the immunodominant B-cell region recognized by human sera [#12]","Source of the H-2Ld-restricted immunodominant CD8+ T cell epitope HF10, immunodominance set by C-terminal epitope position [#3, #5]","GRA6 polymorphism at the C-terminus underlies strain-differential NFAT4 activation (type I > type II) [#6, #10]","Membrane topology (PV membrane insertion vs IVN association) regulates MHC I antigen accessibility [#7]","Stage-specific secretion: secreted in tachyzoites but retained in sporozoite cytoplasmic granules [#11]","Promoter activity mapped to -146 to -27 bp with repeated A/TGAGACG cis-elements [#9]"]},"strain_conflicts":[{"aspect":"Host NFAT4 activation potency driven by GRA6 C-terminal polymorphism","claims":[{"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}],"claim":"Type I parasites cause significantly higher GRA6-dependent NFAT4 activation","supporting_discovery_ids":[6]},{"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}],"claim":"Type II parasites cause substantially lower NFAT4 activation due to a C-terminal GRA6 polymorphism","supporting_discovery_ids":[6]}]}]},"prefetch_data":{},"corpus_meta":[{"pmid":"17963770","id":"PMC_17963770","title":"Population structure and mouse-virulence of Toxoplasma gondii in Brazil.","date":"2007","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/17963770","citation_count":278,"is_preprint":false},{"pmid":"21320505","id":"PMC_21320505","title":"Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North 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The gene encodes a 230-amino-acid polypeptide with two hydrophobic transmembrane-like domains; the N-terminal domain lacks classical signal peptide features, and the C-terminal hydrophilic region is glycine-rich (24%), suggesting a structural role in the network.\",\n      \"method\": \"cDNA cloning, gene sequencing, immunolocalization with specific antiserum (immunofluorescence and immunoelectron microscopy), primer extension analysis\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct subcellular localization by immunoelectron microscopy combined with gene/protein characterization; foundational characterization paper replicated by subsequent studies\",\n      \"pmids\": [\"7637717\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Following secretion into the parasitophorous vacuole, GRA6 is rapidly translocated to the posterior end of the parasite where it binds to a cluster of multi-lamellar vesicles that give rise to the intravacuolar network. GRA6 association with network membranes is predominantly mediated by hydrophobic interactions, and a phosphorylated form of GRA6 shows increased membrane association. Cross-linking studies established that GRA4 and GRA6 specifically interact with GRA2 to form a multimeric complex stably associated with the intravacuolar network.\",\n      \"method\": \"Cell fractionation, denaturing agent treatment, cross-linking studies, immunofluorescence microscopy, immunoelectron microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical methods (fractionation, cross-linking, denaturing agents) in a single rigorous study establishing both localization and protein-protein interactions\",\n      \"pmids\": [\"10498186\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"GRA6 is required for the normal nanotubular conformation of the intravacuolar network (IVN). Deletion of GRA6 (Δgra6 mutant) leads to an altered mature network characterized by small vesicles instead of elongated nanotubules; however, the initial formation of the posterior organizing center was normal in Δgra6 parasites (in contrast to Δgra2 parasites). GRA6 also failed to localize properly to the posterior organizing center in Δgra2 cells, indicating GRA2-dependent localization of GRA6.\",\n      \"method\": \"Gene knockout (Δgra6 and Δgra2 mutants), immunofluorescence microscopy, electron microscopy, complementation with mutated GRA2 forms\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — specific gene knockout with defined ultrastructural phenotype, epistasis analysis with Δgra2, complementation experiments with mutagenized constructs\",\n      \"pmids\": [\"12134078\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GRA6 is the source of the immunodominant and protective decapeptide HF10 presented by the H-2L(d) MHC class I molecule in BALB/c mice. Presentation of the HF10-H-2L(d) ligand required proteolysis by ERAAP (the endoplasmic reticulum aminopeptidase associated with antigen processing), and expansion of protective CD8+ T cell populations was impaired in ERAAP-deficient mice infected with T. gondii.\",\n      \"method\": \"Peptide identification from infected cells, T cell assays, ERAAP knockout mouse infection, CD8+ T cell expansion assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct identification of the epitope source, genetic knockout of processing enzyme (ERAAP) with defined immunological phenotype, in vivo protection assay\",\n      \"pmids\": [\"18587399\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GRA6 (together with GRA4) plays a functional role in brain cyst development and latent infection in type II T. gondii. Deletion of GRA6 in type II Δku80 parasites drastically reduced brain tissue cyst burdens in mice. Complementation with a functional GRA6 allele at the endogenous UPRT locus significantly restored cyst burdens, confirming the specific role of GRA6.\",\n      \"method\": \"Gene knockout in type II Δku80 background, mouse infection model with brain cyst counting, genetic complementation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — specific gene deletion with defined in vivo phenotype, genetic complementation restoring the phenotype, rigorous controls\",\n      \"pmids\": [\"21531875\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru (type II)\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The location of the GRA6 peptide epitope at the C-terminus of the GRA6 antigenic precursor determines immunodominance and optimal MHC I processing. Parasite burden is controlled by GRA6-specific CD8 T cells but not by responses to subdominant GRA4- and ROP7-derived epitopes. Optimal processing and immunodominance were determined by the C-terminal position of the epitope, not by peptide affinity for MHC I or precursor T cell frequency.\",\n      \"method\": \"Genetically modified parasites expressing epitope-repositioned GRA6 variants, in vivo CD8 T cell response assays, parasite burden measurements\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetically modified parasites with epitope repositioning, multiple in vivo readouts, direct functional comparison of dominant vs subdominant responses\",\n      \"pmids\": [\"23818852\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The polymorphic dense granule protein GRA6 selectively activates the host transcription factor NFAT4 via CAMLG. GRA6 overexpression robustly and selectively activated NFAT4. Infection with wild-type but not GRA6-deficient parasites induced NFAT4 activation. GRA6-deficient parasites exhibited reduced virulence in local infection. NFAT4-deficient mice showed prolonged survival, decreased CD11b+Ly6G+ cell recruitment, and impaired expression of chemokines Cxcl2 and Ccl2. Type I parasites caused significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C-terminus of GRA6.\",\n      \"method\": \"GRA6 overexpression, GRA6 gene knockout, NFAT4 knockout mice, in vivo infection assays, flow cytometry, chemokine expression analysis, NFAT inhibitor treatment\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic tools (overexpression, knockout parasite, host knockout), in vivo functional readouts, identification of CAMLG as the molecular intermediary, strain-specific polymorphism linked to activity\",\n      \"pmids\": [\"25225460\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA6 membrane association in the parasitophorous vacuole regulates its accessibility to the MHC class I antigen presentation pathway. Using a cell-free minimal system, GRA6 and GRA2 were shown to shape membrane tubules. Insertion of GRA6 in the PV membrane is key for immunogenicity, whereas association of GRA6 with the IVN limits MHC I presentation and curtails GRA6-specific CD8 T cell responses in mice.\",\n      \"method\": \"Cell-free minimal membrane system reconstitution, MHC I presentation assays in infected cells, CD8 T cell response quantification in mice, GRA6/GRA2 knockout parasites\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free reconstitution of membrane tubule formation, combined with in cellulo antigen presentation assays and in vivo T cell responses, multiple orthogonal methods\",\n      \"pmids\": [\"26628378\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"GRA6 (together with GRA2) contributes to host vesicle sequestration within the parasitophorous vacuole. Rab11A vesicles concentrate at the intravacuolar network (IVN), but this concentration is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that IVN tubules stabilized by GRA2 and GRA6 contribute to host vesicle sequestration.\",\n      \"method\": \"Genetic knockout (Δgra2, Δgra2Δgra6), correlative light and electron microscopy, live imaging of GFP-Rab11A vesicles\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double-knockout with defined vesicle sequestration phenotype, but the Δgra6 single knockout effect on Rab11A was not directly tested alone; conclusion on GRA6 is partly inferred from double-KO\",\n      \"pmids\": [\"29070609\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The promoter of GRA6 contains cis-acting elements critical for expression. Deletion analysis mapped optimal GRA6 promoter activity to a region -146 to -27 bp upstream of the transcription start site. Repeated motifs (A/TGAGACG) found in either orientation within these proximal regions are critical elements for promoter activity of GRA6 (and other GRA genes).\",\n      \"method\": \"Transient transformation with CAT reporter gene constructs, deletion analysis of promoter regions, mutation analysis of cis-elements\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter gene assays with deletion and mutation analysis in a single lab, defines promoter elements functionally\",\n      \"pmids\": [\"8858595\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"GRA6 sequence polymorphisms correlate with T. gondii strain type and murine virulence. Sequence alignment of the GRA6 coding region (690 bp) across 30 strains from 8 zymodemes identified 24 polymorphic nucleotide positions distinguishing types I, II, and III. Type II strains have unique deletions of 15 bp and 3 bp causing loss of 6 amino acids. A PCR-RFLP method using MseI was developed to distinguish the three lineages based on GRA6 polymorphisms.\",\n      \"method\": \"PCR amplification and DNA sequencing of GRA6 coding region from 30 strains, sequence alignment, PCR-RFLP with MseI enzyme\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — sequence-based characterization across multiple strains establishing genotype-phenotype correlation, but mechanism is correlational rather than experimentally dissected\",\n      \"pmids\": [\"10779578\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"In sporozoites, GRA6 protein is present in dense granules and is detected in oocyst extracts, but unlike tachyzoite behavior, GRA6 (along with GRA1, GRA2, GRA4) is retained within cytoplasmic granules during sporozoite invasion of host cells and is not secreted into the first parasitophorous vacuole (PV1). This establishes a stage-specific difference in GRA6 secretion behavior.\",\n      \"method\": \"Immunofluorescence with anti-GRA antibodies on sporozoite-infected cells, comparison of tachyzoite vs sporozoite secretion, oocyst extract immunoblot\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — immunolocalization studies comparing two life stages, single lab but with multiple antibodies and consistent results\",\n      \"pmids\": [\"9353039\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The N-terminal hydrophilic region of GRA6 (but not the C-terminal region) is recognized by human anti-Toxoplasma sera. Recombinant GST-GRA6-Nt ELISA achieved 96% sensitivity for detection of T. gondii IgG in human sera, demonstrating that the N-terminal hydrophilic domain is the immunodominant region of GRA6 in humans.\",\n      \"method\": \"Recombinant protein expression in E. coli (separate N- and C-terminal domains), immunoblot with pooled human sera, ELISA with 100 positive and 98 negative human sera\",\n      \"journal\": \"Clinical and diagnostic laboratory immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain-mapping by differential recombinant protein reactivity with a well-characterized serum panel, single lab\",\n      \"pmids\": [\"10882660\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GRA12 behaves similarly to GRA6 in that it is secreted into the PV from the anterior pole, transits to the posterior invagination where the IVN assembles, and finally resides in the vacuolar space associated with the mature nanotubular network. GRA12 fails to localize at the parasite posterior end in the absence of GRA2, similar to GRA6. Affinity chromatography using tagged GRA2 co-purified GRA12 with GRA2 in both parasite and PV soluble fractions, placing GRA12 in the same GRA protein complex that includes GRA6.\",\n      \"method\": \"Immunofluorescence, immunoelectron microscopy, GRA2 knockout analysis, affinity chromatography with tagged GRA2\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — GRA12 characterization paper that provides additional evidence for GRA6 complex membership via affinity chromatography; GRA6 is a comparator, not the primary subject\",\n      \"pmids\": [\"18840447\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"GRA6 is a dense granule protein secreted into the Toxoplasma gondii parasitophorous vacuole where it associates with the intravacuolar network (IVN) via hydrophobic interactions and a GRA2-dependent posterior organizing center; it stabilizes IVN nanotubular architecture, facilitates host Rab vesicle sequestration, forms a multimeric complex with GRA2 and GRA4, promotes brain cyst development during chronic infection, and acts as a host-signaling effector by activating NFAT4 through CAMLG in a strain-polymorphism-dependent manner, while also serving as a source of the immunodominant CD8+ T cell epitope HF10 whose access to MHC I presentation is regulated by GRA6 membrane topology within the PV.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA6 is a dense granule protein of Toxoplasma gondii that is secreted into the parasitophorous vacuole (PV), where it functions both as a structural organizer of the intravacuolar network (IVN) and as a strain-polymorphic effector of host signaling and antigen presentation [#0, #6, #7]. After secretion from the anterior pole, GRA6 is translocated to the posterior end of the parasite and associates with the nascent IVN membranes predominantly through hydrophobic interactions, forming a stable multimeric complex with GRA2 and GRA4 [#1]. Its correct posterior localization depends on GRA2, and GRA6 is in turn required for the mature nanotubular conformation of the IVN, since Δgra6 vacuoles accumulate small vesicles rather than elongated tubules [#2]; in a reconstituted cell-free system GRA6 and GRA2 are sufficient to shape membrane tubules [#7]. This GRA2/GRA6-stabilized network contributes to sequestration of host Rab11A vesicles within the PV [#8] and promotes brain tissue cyst development during chronic type II infection [#4]. GRA6 also acts as a host-manipulating effector, selectively activating the transcription factor NFAT4 through CAMLG in a manner dependent on a C-terminal polymorphism that makes type I strains far more potent than type II, driving myeloid cell recruitment and chemokine induction [#6]. Independently, GRA6 supplies the immunodominant, ERAAP-dependent decapeptide HF10 presented by H-2Ld to protective CD8+ T cells, with immunodominance dictated by the C-terminal position of the epitope [#3, #5], and epitope accessibility governed by GRA6 membrane topology—PV-membrane insertion favors presentation whereas IVN association limits it [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing that GRA6 is a dense granule antigen that relocates to the intravacuolar network defined it as a secreted structural component of the PV rather than a purely cytosolic parasite protein.\",\n      \"evidence\": \"cDNA cloning/sequencing plus immunofluorescence and immunoelectron microscopy localization\",\n      \"pmids\": [\"7637717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the two hydrophobic domains and glycine-rich C-terminus not tested\", \"No interacting partners identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Mapping cis-acting promoter elements answered how GRA6 expression is transcriptionally controlled, identifying repeated A/TGAGACG motifs shared with other GRA genes.\",\n      \"evidence\": \"CAT reporter deletion and mutation analysis in transient transformation\",\n      \"pmids\": [\"8858595\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trans-acting factors binding these motifs not identified\", \"Regulation across life stages not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Comparing life stages revealed that GRA6 secretion behavior is stage-specific, being retained in cytoplasmic granules during sporozoite invasion rather than secreted into the vacuole.\",\n      \"evidence\": \"Immunofluorescence and immunoblot comparing sporozoite versus tachyzoite behavior\",\n      \"pmids\": [\"9353039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of stage-specific retention unknown\", \"Consequence for sporozoite PV biology untested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Biochemical dissection of GRA6 membrane association showed it binds IVN membranes via hydrophobic interactions and forms a defined complex with GRA2 and GRA4, moving from localization to a molecular assembly model.\",\n      \"evidence\": \"Cell fractionation, denaturing treatment, cross-linking, and electron microscopy\",\n      \"pmids\": [\"10498186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and interface of the GRA2/GRA4/GRA6 complex not resolved\", \"Role of GRA6 phosphorylation not functionally defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Sequencing GRA6 across strains established a genotype-phenotype correlation, linking GRA6 polymorphisms to strain lineage and murine virulence and providing a genotyping tool.\",\n      \"evidence\": \"PCR/sequencing of GRA6 across 30 strains and PCR-RFLP with MseI\",\n      \"pmids\": [\"10779578\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Correlation is not a demonstrated causal mechanism\", \"Functional consequence of specific polymorphisms not tested here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Domain mapping of the human antibody response identified the N-terminal hydrophilic region as the immunodominant B-cell region of GRA6, informing serodiagnostic use.\",\n      \"evidence\": \"Reactivity of recombinant N- versus C-terminal domains with human sera by immunoblot and ELISA\",\n      \"pmids\": [\"10882660\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address T-cell epitope location\", \"Single serum panel\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Knockout epistasis established that GRA6 is required for the mature nanotubular architecture of the IVN and that its posterior localization is GRA2-dependent, defining a hierarchy within the network-building machinery.\",\n      \"evidence\": \"Δgra6 and Δgra2 knockouts, EM/IF ultrastructural analysis, complementation with mutant GRA2\",\n      \"pmids\": [\"12134078\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GRA6 remodels vesicles into tubules not resolved at this stage\", \"Consequence of altered IVN for parasite fitness not measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying GRA6 as the source of the ERAAP-processed HF10/H-2Ld epitope explained a major immunodominant CD8+ T cell response and linked antigen processing machinery to protective immunity.\",\n      \"evidence\": \"Epitope identification, T cell assays, and ERAAP-knockout mouse infection\",\n      \"pmids\": [\"18587399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why GRA6-derived HF10 is preferentially processed not yet explained\", \"Restricted to BALB/c H-2Ld context\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Characterization of GRA12 showed it co-purifies with GRA2 and shares GRA6's trafficking route, extending the composition of the GRA2-anchored IVN complex that includes GRA6.\",\n      \"evidence\": \"IF/immunoEM, GRA2-knockout analysis, and affinity chromatography with tagged GRA2\",\n      \"pmids\": [\"18840447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct GRA6-GRA12 interaction not demonstrated\", \"GRA6 is a comparator rather than the primary subject\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Deletion and complementation in type II parasites established that GRA6 promotes brain cyst development, linking an IVN structural protein to chronic latent infection.\",\n      \"evidence\": \"Δgra6 in type II Δku80, mouse brain cyst counting, and complementation at the UPRT locus\",\n      \"pmids\": [\"21531875\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the cyst defect reflects IVN disruption or another GRA6 activity is unresolved\", \"Molecular pathway to cyst formation not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru (type II)\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Epitope-repositioning experiments showed that GRA6 immunodominance is dictated by the C-terminal position of HF10, not by MHC affinity or precursor frequency, defining a positional rule for antigen presentation and protection.\",\n      \"evidence\": \"Epitope-repositioned GRA6 variant parasites, in vivo CD8 T cell responses, and parasite burden measurement\",\n      \"pmids\": [\"23818852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis of the C-terminal positional advantage not fully defined\", \"Generality to other GRA epitopes untested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that GRA6 selectively activates NFAT4 via CAMLG in a strain-polymorphism-dependent manner recast GRA6 as a host-signaling effector controlling immune cell recruitment and virulence.\",\n      \"evidence\": \"GRA6 overexpression and knockout, NFAT4-knockout mice, chemokine analysis, and NFAT inhibitor treatment\",\n      \"pmids\": [\"25225460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PV-resident GRA6 engages host CAMLG topologically not resolved\", \"Precise polymorphic residues driving type I vs type II potency not enumerated here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Cell-free reconstitution and presentation assays unified GRA6 structural and immunological roles, showing GRA6/GRA2 shape membrane tubules and that GRA6 membrane topology (PV membrane insertion vs IVN association) gates its MHC I accessibility.\",\n      \"evidence\": \"Cell-free minimal membrane system, MHC I presentation assays, in vivo CD8 T cell responses, GRA6/GRA2 knockouts\",\n      \"pmids\": [\"26628378\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What controls partitioning of GRA6 between PV membrane and IVN in vivo is unresolved\", \"Link between topology and NFAT4 signaling not directly tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Double-knockout imaging tied the GRA2/GRA6-stabilized IVN to host vesicle sequestration, connecting network architecture to nutrient/host-vesicle acquisition.\",\n      \"evidence\": \"Δgra2 and Δgra2Δgra6 parasites, correlative light-electron microscopy, and live GFP-Rab11A imaging\",\n      \"pmids\": [\"29070609\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Δgra6 single-knockout effect on Rab11A not tested alone\", \"Functional consequence of Rab11A sequestration for the parasite not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRA6 is partitioned between the PV membrane and the IVN in vivo, and how this topology mechanistically couples its structural, host-signaling (NFAT4/CAMLG), and antigen-presentation functions, remains unresolved.\",\n      \"evidence\": \"No direct experimental resolution in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Regulators of GRA6 membrane topology unknown\", \"Structural interface of the GRA2/GRA4/GRA6 complex undetermined\", \"Direct GRA6-CAMLG interaction and its topological requirement not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [2, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0042803\", \"supporting_discovery_ids\": [1, 13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0, 1, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020026\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0002479\", \"supporting_discovery_ids\": [3, 5, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051092\", \"supporting_discovery_ids\": [6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0044406\", \"supporting_discovery_ids\": [4, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"GRA2-GRA4-GRA6 multimeric intravacuolar network complex\",\n      \"GRA2-associated IVN complex including GRA12\"\n    ],\n    \"partners\": [\n      \"GRA2\",\n      \"GRA4\",\n      \"GRA12 (co-complex via GRA2)\",\n      \"CAMLG (host)\",\n      \"NFAT4 (host, activated downstream)\",\n      \"ERAAP (host antigen-processing aminopeptidase)\"\n    ],\n    \"other_free_text\": [\n      \"230-aa polypeptide with two hydrophobic transmembrane-like domains and a glycine-rich C-terminal hydrophilic region [#0]\",\n      \"N-terminal hydrophilic region is the immunodominant B-cell region recognized by human sera [#12]\",\n      \"Source of the H-2Ld-restricted immunodominant CD8+ T cell epitope HF10, immunodominance set by C-terminal epitope position [#3, #5]\",\n      \"GRA6 polymorphism at the C-terminus underlies strain-differential NFAT4 activation (type I > type II) [#6, #10]\",\n      \"Membrane topology (PV membrane insertion vs IVN association) regulates MHC I antigen accessibility [#7]\",\n      \"Stage-specific secretion: secreted in tachyzoites but retained in sporozoite cytoplasmic granules [#11]\",\n      \"Promoter activity mapped to -146 to -27 bp with repeated A/TGAGACG cis-elements [#9]\"\n    ]\n  },\n  \"strain_conflicts\": [\n    {\n      \"aspect\": \"Host NFAT4 activation potency driven by GRA6 C-terminal polymorphism\",\n      \"claims\": [\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}], \"claim\": \"Type I parasites cause significantly higher GRA6-dependent NFAT4 activation\", \"supporting_discovery_ids\": [6]},\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}], \"claim\": \"Type II parasites cause substantially lower NFAT4 activation due to a C-terminal GRA6 polymorphism\", \"supporting_discovery_ids\": [6]}\n      ]\n    }\n  ]\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_275440","name":"GRA6","product":"dense granule protein GRA6","uniprot_acc":"A0A125YS55","chromosome":"TGME49_chrX","start_pos":7270768},{"strain":"GT1","gene_id":"TGGT1_275440","name":null,"product":"dense granule protein GRA6","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":7158307},{"strain":"RH88","gene_id":"TGRH88_038710","name":null,"product":"dense granule protein GRA6","uniprot_acc":null,"chromosome":"CM023091","start_pos":261651},{"strain":"VEG","gene_id":"TGVEG_275440","name":null,"product":"dense granule protein GRA6","uniprot_acc":"A0A0F7V3T7","chromosome":"TGVEG_chrX","start_pos":7193731}]}