{"gene":"TG_226380","gene_symbol":"GRA35","run_date":"2026-09-05T22:31:42","timeline":{"discoveries":[{"year":2019,"finding":"GRA35, GRA42, and GRA43 are individually required for induction of Lewis rat macrophage pyroptosis; deletion of any single GRA abrogates cell death and enhances parasite replication, and single/double/triple deletions show similar cell viability, indicating these three proteins function in the same pathway.","method":"Chemical mutagenesis screen, whole-genome sequencing, targeted gene deletion (Δgra35, Δgra42, Δgra43), macrophage infection assays","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple independent knockout lines with defined cellular phenotype (pyroptosis, replication), epistasis analysis across single/double/triple deletions, replicated across conditions","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GRA35 localizes to the parasitophorous vacuole membrane (PVM), but deletion of GRA42 or GRA43 causes GRA35 (and other GRAs) to be retained inside the parasitophorous vacuole rather than reaching the PVM, demonstrating that GRA42 and GRA43 are required for correct GRA35 localization to the PVM.","method":"Gene deletion, immunofluorescence/localization assays of GRA35 in Δgra42 and Δgra43 parasites","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment with functional consequence, multiple knockout backgrounds tested, single lab with multiple orthogonal methods","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GRA35, GRA42, and GRA43 are required for parasite in vivo fitness in F344 rats (reducing cyst numbers when deleted), independently of their role in NLRP1 inflammasome/pyroptosis activation, indicating an additional function in chronic infection establishment.","method":"Gene deletion, in vivo rat infection, cyst burden quantification","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout with defined in vivo phenotype, single lab, genetic evidence for pathway independence","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"GRA35 localizes on the parasitophorous vacuole membrane where it directly interacts with the host E3 ubiquitin ligase ITCH; this interaction is required for NLRP1 inflammasome activation and macrophage pyroptosis in Lewis rats, consistent with the NLRP1 functional degradation model (proteasome inhibition or ITCH knockout prevented pyroptosis).","method":"Genome-wide CRISPR screen, localization assays (immunofluorescence), protein interaction (Co-IP/pulldown implied), ITCH knockout, proteasome inhibitor experiments","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide CRISPR screen identifying GRA35, direct interaction with ITCH demonstrated, genetic knockout of ITCH confirming functional requirement, multiple orthogonal methods, peer-reviewed publication","pmids":["38376248"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"The GRA35-ITCH interaction also affects Toxoplasma fitness in IFNγ-activated human fibroblasts, likely because ITCH recruits ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane.","method":"IFNγ-activated human fibroblast infection assays, ITCH knockout, RNF213 localization assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined fitness phenotype in knockout cells with mechanistic localization data, single lab, single publication","pmids":["38376248"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"The same mechanistic findings (GRA35 on PVM interacting with ITCH to mediate NLRP1 pyroptosis; no evidence ITCH directly ubiquitinates rat NLRP1) were reported as a preprint prior to peer-reviewed publication.","method":"Genome-wide CRISPR screen, protein interaction, ITCH knockout, proteasome inhibition","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — preprint version of peer-reviewed study; same data, included for completeness but superseded by published version","pmids":["38168400"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"GRA35 interacts with RTN1-c (reticulon 1-c) and promotes mitochondrial pathway-mediated apoptosis in neurons; GRA35 also triggers ER stress-associated apoptosis through the PERK signaling pathway. Deletion of GRA35 in the ME49 strain reduces neuronal apoptosis in the brain during chronic infection.","method":"GRA35 knockout in ME49 strain, GST pulldown (GRA35–RTN1-c interaction), immunofluorescence, flow cytometry, CCK8 assay","journal":"Virulence","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — GST pulldown identifies RTN1-c as binding partner, knockout phenotype confirmed in brain neurons, single lab, single publication","pmids":["41940502"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]}],"current_model":"GRA35 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) in a manner dependent on GRA42 and GRA43; at the PVM, GRA35 interacts with the host E3 ubiquitin ligase ITCH to activate the NLRP1 inflammasome and trigger pyroptosis in Lewis rat macrophages, while also recruiting ubiquitin and RNF213 to restrict parasite fitness in IFNγ-activated human fibroblasts; additionally, GRA35 promotes neuronal apoptosis by binding RTN1-c (activating the mitochondrial apoptosis pathway) and by inducing ER stress via PERK signaling, collectively establishing GRA35 as a multifunctional effector at the parasite–host interface."},"narrative":{"mechanistic_narrative":"GRA35 is a Toxoplasma gondii dense granule protein that acts as a multifunctional effector at the parasite–host interface, engaging host cell death and immune restriction pathways from the parasitophorous vacuole membrane (PVM) [PMID:30622189, PMID:38376248]. Its correct trafficking to the PVM requires GRA42 and GRA43, and loss of either retains GRA35 within the parasitophorous vacuole; all three proteins function in a common pathway required to induce Lewis rat macrophage pyroptosis [PMID:30622189]. At the PVM, GRA35 directly binds the host E3 ubiquitin ligase ITCH, an interaction required for NLRP1 inflammasome activation and pyroptosis consistent with a proteasome-dependent NLRP1 functional-degradation model [PMID:38376248]. The same GRA35–ITCH interaction restricts parasite fitness in IFNγ-activated human fibroblasts by recruiting ubiquitin and the restriction factor RNF213 to the PVM [PMID:38376248]. In ME49 chronic brain infection, GRA35 additionally promotes neuronal apoptosis by binding RTN1-c to engage the mitochondrial apoptosis pathway and by inducing PERK-mediated ER stress [PMID:41940502]. Beyond its role in pyroptosis, GRA35 (together with GRA42 and GRA43) contributes to in vivo fitness and chronic cyst establishment in rats independently of inflammasome activation [PMID:30622189].","teleology":[{"year":2019,"claim":"Establishing which parasite factors drive host macrophage pyroptosis, a forward-genetic screen defined GRA35, GRA42, and GRA43 as components of a single pathway required for cell death and replication control.","evidence":"Chemical mutagenesis screen with whole-genome sequencing, targeted single/double/triple deletions, and macrophage infection assays in Lewis rats","pmids":["30622189"],"confidence":"High","gaps":["Molecular event triggering pyroptosis downstream of the three GRAs was not defined","Direct host binding partner of GRA35 not yet identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"To place the three GRAs in a spatial hierarchy, localization studies showed GRA42 and GRA43 are required to deliver GRA35 to the PVM, establishing them as trafficking determinants for GRA35 function.","evidence":"Immunofluorescence localization of GRA35 in Δgra42 and Δgra43 parasites","pmids":["30622189"],"confidence":"High","gaps":["Mechanism by which GRA42/GRA43 mediate PVM delivery unknown","Whether GRA42/GRA43 act during translocation or membrane insertion not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Testing whether the GRAs matter beyond acute cell death, in vivo infection revealed they support chronic cyst establishment independently of inflammasome/pyroptosis activation.","evidence":"Gene deletion and cyst burden quantification in F344 rats","pmids":["30622189"],"confidence":"Medium","gaps":["Molecular basis of the pyroptosis-independent fitness role unknown","Host pathway underlying reduced cyst numbers not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Identifying the host receptor for GRA35's pyroptotic activity, an unbiased CRISPR screen and interaction studies showed GRA35 directly binds the E3 ubiquitin ligase ITCH to activate the NLRP1 inflammasome via a proteasome-dependent functional-degradation mechanism.","evidence":"Genome-wide CRISPR screen, immunofluorescence, Co-IP/pulldown, ITCH knockout, and proteasome inhibitor experiments in Lewis rat cells","pmids":["38376248","38168400"],"confidence":"High","gaps":["No evidence that ITCH directly ubiquitinates rat NLRP1","Structural basis of the GRA35–ITCH interaction unresolved","How ITCH engagement couples to NLRP1 degradation not fully mechanistically defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Extending the GRA35–ITCH axis to cell-autonomous immunity, the interaction was shown to reduce parasite fitness in IFNγ-activated human fibroblasts by recruiting ubiquitin and the restriction factor RNF213 to the PVM.","evidence":"IFNγ-activated human fibroblast infection assays, ITCH knockout, and RNF213 localization assays","pmids":["38376248"],"confidence":"Medium","gaps":["Direct causal link between RNF213 recruitment and restriction not fully established","Whether GRA35 confers advantage or liability across cell types not reconciled"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"Probing GRA35's role in chronic CNS infection, GRA35 was shown to bind RTN1-c and drive neuronal apoptosis via both the mitochondrial pathway and PERK-mediated ER stress.","evidence":"GRA35 knockout in ME49, GST pulldown (GRA35–RTN1-c), immunofluorescence, flow cytometry, and CCK8 assays in brain neurons","pmids":["41940502"],"confidence":"Medium","gaps":["GST pulldown interaction not validated by reciprocal or in vivo methods","How PVM-localized GRA35 accesses neuronal RTN1-c and ER machinery not defined","Relationship between apoptosis induction and inflammasome/ITCH functions unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":null,"claim":"How GRA35's distinct host-directed activities—NLRP1/pyroptosis, ubiquitin/RNF213 restriction, and RTN1-c/PERK-driven neuronal apoptosis—are integrated at the domain level and coordinated across host cell types and infection stages remains unknown.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or domain-mapping data linking the multiple binding activities of GRA35","No unified model reconciling parasite-protective versus parasite-restrictive outcomes"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["ITCH (HOST E3 UBIQUITIN LIGASE)","RTN1-C (RETICULON 1-C)","GRA42 (TRAFFICKING DEPENDENCY)","GRA43 (TRAFFICKING DEPENDENCY)","RNF213 (RECRUITED RESTRICTION FACTOR)"],"other_free_text":["Dense granule protein trafficked to the PVM in a GRA42/GRA43-dependent manner [#1].","Activates NLRP1 inflammasome via ITCH consistent with a proteasome-dependent functional-degradation model [#3].","Recruits ubiquitin and RNF213 to the PVM to restrict fitness in IFNγ-activated human fibroblasts [#4].","Contributes to chronic cyst establishment in rats independently of pyroptosis [#2]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"30622189","id":"PMC_30622189","title":"Three Toxoplasma gondii Dense Granule Proteins Are Required for Induction of Lewis Rat Macrophage Pyroptosis.","date":"2019","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/30622189","citation_count":63,"is_preprint":false},{"pmid":"38029261","id":"PMC_38029261","title":"Protective immunity induced by DNA vaccine containing TgGRA35, TgGRA42, and TgGRA43 against Toxoplasma gondii infection in Kunming mice.","date":"2023","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/38029261","citation_count":13,"is_preprint":false},{"pmid":"38376248","id":"PMC_38376248","title":"Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.","date":"2024","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/38376248","citation_count":11,"is_preprint":false},{"pmid":"38310017","id":"PMC_38310017","title":"Protective effect against toxoplasmosis in BALB/C mice vaccinated with recombinant Toxoplasma gondii CDPK3, GRA35, and ROP46 protein cocktail vaccine.","date":"2024","source":"Vaccine","url":"https://pubmed.ncbi.nlm.nih.gov/38310017","citation_count":4,"is_preprint":false},{"pmid":"41296383","id":"PMC_41296383","title":"Development and Evaluation of Six Novel Recombinant GRA Proteins in Serodiagnosis of Human Toxoplasmosis.","date":"2025","source":"Current issues in molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/41296383","citation_count":2,"is_preprint":false},{"pmid":"37287466","id":"PMC_37287466","title":"Variation in CD8 T cell IFNγ differentiation to strains of Toxoplasma gondii is characterized by small effect QTLs with contribution from ROP16.","date":"2023","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/37287466","citation_count":1,"is_preprint":false},{"pmid":"41940502","id":"PMC_41940502","title":"Toxoplasma gondii effector GRA35 mediates neuronal damage via ER stress and mitochondria-associated apoptosis.","date":"2026","source":"Virulence","url":"https://pubmed.ncbi.nlm.nih.gov/41940502","citation_count":0,"is_preprint":false},{"pmid":"38168400","id":"PMC_38168400","title":"Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38168400","citation_count":0,"is_preprint":false},{"pmid":"42197386","id":"PMC_42197386","title":"Heterologous Prime-Boost Vaccination with GRA35-Encoding DNA and mRNA Vaccines Enhances Protective Immunity Against Toxoplasma gondii Infection in Mouse Models.","date":"2026","source":"Microorganisms","url":"https://pubmed.ncbi.nlm.nih.gov/42197386","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7750,"output_tokens":1938,"usd":0.02616,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7315,"output_tokens":3195,"usd":0.058225,"stage2_stop_reason":"end_turn"},"total_usd":0.084385,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA35, GRA42, and GRA43 are individually required for induction of Lewis rat macrophage pyroptosis; deletion of any single GRA abrogates cell death and enhances parasite replication, and single/double/triple deletions show similar cell viability, indicating these three proteins function in the same pathway.\",\n      \"method\": \"Chemical mutagenesis screen, whole-genome sequencing, targeted gene deletion (Δgra35, Δgra42, Δgra43), macrophage infection assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple independent knockout lines with defined cellular phenotype (pyroptosis, replication), epistasis analysis across single/double/triple deletions, replicated across conditions\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA35 localizes to the parasitophorous vacuole membrane (PVM), but deletion of GRA42 or GRA43 causes GRA35 (and other GRAs) to be retained inside the parasitophorous vacuole rather than reaching the PVM, demonstrating that GRA42 and GRA43 are required for correct GRA35 localization to the PVM.\",\n      \"method\": \"Gene deletion, immunofluorescence/localization assays of GRA35 in Δgra42 and Δgra43 parasites\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment with functional consequence, multiple knockout backgrounds tested, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA35, GRA42, and GRA43 are required for parasite in vivo fitness in F344 rats (reducing cyst numbers when deleted), independently of their role in NLRP1 inflammasome/pyroptosis activation, indicating an additional function in chronic infection establishment.\",\n      \"method\": \"Gene deletion, in vivo rat infection, cyst burden quantification\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with defined in vivo phenotype, single lab, genetic evidence for pathway independence\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GRA35 localizes on the parasitophorous vacuole membrane where it directly interacts with the host E3 ubiquitin ligase ITCH; this interaction is required for NLRP1 inflammasome activation and macrophage pyroptosis in Lewis rats, consistent with the NLRP1 functional degradation model (proteasome inhibition or ITCH knockout prevented pyroptosis).\",\n      \"method\": \"Genome-wide CRISPR screen, localization assays (immunofluorescence), protein interaction (Co-IP/pulldown implied), ITCH knockout, proteasome inhibitor experiments\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide CRISPR screen identifying GRA35, direct interaction with ITCH demonstrated, genetic knockout of ITCH confirming functional requirement, multiple orthogonal methods, peer-reviewed publication\",\n      \"pmids\": [\"38376248\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The GRA35-ITCH interaction also affects Toxoplasma fitness in IFNγ-activated human fibroblasts, likely because ITCH recruits ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane.\",\n      \"method\": \"IFNγ-activated human fibroblast infection assays, ITCH knockout, RNF213 localization assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined fitness phenotype in knockout cells with mechanistic localization data, single lab, single publication\",\n      \"pmids\": [\"38376248\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The same mechanistic findings (GRA35 on PVM interacting with ITCH to mediate NLRP1 pyroptosis; no evidence ITCH directly ubiquitinates rat NLRP1) were reported as a preprint prior to peer-reviewed publication.\",\n      \"method\": \"Genome-wide CRISPR screen, protein interaction, ITCH knockout, proteasome inhibition\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — preprint version of peer-reviewed study; same data, included for completeness but superseded by published version\",\n      \"pmids\": [\"38168400\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GRA35 interacts with RTN1-c (reticulon 1-c) and promotes mitochondrial pathway-mediated apoptosis in neurons; GRA35 also triggers ER stress-associated apoptosis through the PERK signaling pathway. Deletion of GRA35 in the ME49 strain reduces neuronal apoptosis in the brain during chronic infection.\",\n      \"method\": \"GRA35 knockout in ME49 strain, GST pulldown (GRA35–RTN1-c interaction), immunofluorescence, flow cytometry, CCK8 assay\",\n      \"journal\": \"Virulence\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — GST pulldown identifies RTN1-c as binding partner, knockout phenotype confirmed in brain neurons, single lab, single publication\",\n      \"pmids\": [\"41940502\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    }\n  ],\n  \"current_model\": \"GRA35 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) in a manner dependent on GRA42 and GRA43; at the PVM, GRA35 interacts with the host E3 ubiquitin ligase ITCH to activate the NLRP1 inflammasome and trigger pyroptosis in Lewis rat macrophages, while also recruiting ubiquitin and RNF213 to restrict parasite fitness in IFNγ-activated human fibroblasts; additionally, GRA35 promotes neuronal apoptosis by binding RTN1-c (activating the mitochondrial apoptosis pathway) and by inducing ER stress via PERK signaling, collectively establishing GRA35 as a multifunctional effector at the parasite–host interface.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA35 is a Toxoplasma gondii dense granule protein that acts as a multifunctional effector at the parasite–host interface, engaging host cell death and immune restriction pathways from the parasitophorous vacuole membrane (PVM) [#0, #3]. Its correct trafficking to the PVM requires GRA42 and GRA43, and loss of either retains GRA35 within the parasitophorous vacuole; all three proteins function in a common pathway required to induce Lewis rat macrophage pyroptosis [#0, #1]. At the PVM, GRA35 directly binds the host E3 ubiquitin ligase ITCH, an interaction required for NLRP1 inflammasome activation and pyroptosis consistent with a proteasome-dependent NLRP1 functional-degradation model [#3]. The same GRA35–ITCH interaction restricts parasite fitness in IFNγ-activated human fibroblasts by recruiting ubiquitin and the restriction factor RNF213 to the PVM [#4]. In ME49 chronic brain infection, GRA35 additionally promotes neuronal apoptosis by binding RTN1-c to engage the mitochondrial apoptosis pathway and by inducing PERK-mediated ER stress [#6]. Beyond its role in pyroptosis, GRA35 (together with GRA42 and GRA43) contributes to in vivo fitness and chronic cyst establishment in rats independently of inflammasome activation [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Establishing which parasite factors drive host macrophage pyroptosis, a forward-genetic screen defined GRA35, GRA42, and GRA43 as components of a single pathway required for cell death and replication control.\",\n      \"evidence\": \"Chemical mutagenesis screen with whole-genome sequencing, targeted single/double/triple deletions, and macrophage infection assays in Lewis rats\",\n      \"pmids\": [\"30622189\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular event triggering pyroptosis downstream of the three GRAs was not defined\", \"Direct host binding partner of GRA35 not yet identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"To place the three GRAs in a spatial hierarchy, localization studies showed GRA42 and GRA43 are required to deliver GRA35 to the PVM, establishing them as trafficking determinants for GRA35 function.\",\n      \"evidence\": \"Immunofluorescence localization of GRA35 in Δgra42 and Δgra43 parasites\",\n      \"pmids\": [\"30622189\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GRA42/GRA43 mediate PVM delivery unknown\", \"Whether GRA42/GRA43 act during translocation or membrane insertion not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Testing whether the GRAs matter beyond acute cell death, in vivo infection revealed they support chronic cyst establishment independently of inflammasome/pyroptosis activation.\",\n      \"evidence\": \"Gene deletion and cyst burden quantification in F344 rats\",\n      \"pmids\": [\"30622189\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the pyroptosis-independent fitness role unknown\", \"Host pathway underlying reduced cyst numbers not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying the host receptor for GRA35's pyroptotic activity, an unbiased CRISPR screen and interaction studies showed GRA35 directly binds the E3 ubiquitin ligase ITCH to activate the NLRP1 inflammasome via a proteasome-dependent functional-degradation mechanism.\",\n      \"evidence\": \"Genome-wide CRISPR screen, immunofluorescence, Co-IP/pulldown, ITCH knockout, and proteasome inhibitor experiments in Lewis rat cells\",\n      \"pmids\": [\"38376248\", \"38168400\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No evidence that ITCH directly ubiquitinates rat NLRP1\", \"Structural basis of the GRA35–ITCH interaction unresolved\", \"How ITCH engagement couples to NLRP1 degradation not fully mechanistically defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extending the GRA35–ITCH axis to cell-autonomous immunity, the interaction was shown to reduce parasite fitness in IFNγ-activated human fibroblasts by recruiting ubiquitin and the restriction factor RNF213 to the PVM.\",\n      \"evidence\": \"IFNγ-activated human fibroblast infection assays, ITCH knockout, and RNF213 localization assays\",\n      \"pmids\": [\"38376248\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct causal link between RNF213 recruitment and restriction not fully established\", \"Whether GRA35 confers advantage or liability across cell types not reconciled\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Probing GRA35's role in chronic CNS infection, GRA35 was shown to bind RTN1-c and drive neuronal apoptosis via both the mitochondrial pathway and PERK-mediated ER stress.\",\n      \"evidence\": \"GRA35 knockout in ME49, GST pulldown (GRA35–RTN1-c), immunofluorescence, flow cytometry, and CCK8 assays in brain neurons\",\n      \"pmids\": [\"41940502\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GST pulldown interaction not validated by reciprocal or in vivo methods\", \"How PVM-localized GRA35 accesses neuronal RTN1-c and ER machinery not defined\", \"Relationship between apoptosis induction and inflammasome/ITCH functions unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRA35's distinct host-directed activities—NLRP1/pyroptosis, ubiquitin/RNF213 restriction, and RTN1-c/PERK-driven neuronal apoptosis—are integrated at the domain level and coordinated across host cell types and infection stages remains unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural or domain-mapping data linking the multiple binding activities of GRA35\", \"No unified model reconciling parasite-protective versus parasite-restrictive outcomes\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0019903\", \"supporting_discovery_ids\": [3, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0070269\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006915\", \"supporting_discovery_ids\": [6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]},\n      {\"term_id\": \"GO:0036498\", \"supporting_discovery_ids\": [6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ITCH (host E3 ubiquitin ligase)\",\n      \"RTN1-c (reticulon 1-c)\",\n      \"GRA42 (trafficking dependency)\",\n      \"GRA43 (trafficking dependency)\",\n      \"RNF213 (recruited restriction factor)\"\n    ],\n    \"other_free_text\": [\n      \"Dense granule protein trafficked to the PVM in a GRA42/GRA43-dependent manner [#1].\",\n      \"Activates NLRP1 inflammasome via ITCH consistent with a proteasome-dependent functional-degradation model [#3].\",\n      \"Recruits ubiquitin and RNF213 to the PVM to restrict fitness in IFNγ-activated human fibroblasts [#4].\",\n      \"Contributes to chronic cyst establishment in rats independently of pyroptosis [#2].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_226380","name":"GRA35","product":"dense granule protein GRA35","uniprot_acc":"S8EXL5","chromosome":"TGME49_chrX","start_pos":1675164},{"strain":"GT1","gene_id":"TGGT1_226380","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":1583226},{"strain":"RH88","gene_id":"TGRH88_045940","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023091","start_pos":5896270},{"strain":"VEG","gene_id":"TGVEG_226380","name":null,"product":"putative transmembrane protein","uniprot_acc":"B9Q806","chromosome":"TGVEG_chrX","start_pos":1568048}]}