{"gene":"TG_212300","gene_symbol":"GRA32","run_date":"2026-09-05T22:31:42","timeline":{"discoveries":[{"year":2016,"finding":"GRA32 was identified as a dense granule protein localized to the Toxoplasma parasitophorous vacuole, detected by in vivo BirA*-based proximity biotinylation and affinity purification/mass spectrometry, with localization confirmed by endogenous gene tagging.","method":"In vivo proximity biotinylation (BirA* fusion), affinity purification, mass spectrometry, endogenous gene tagging/localization","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization confirmed by endogenous tagging plus MS identification; single lab, two orthogonal methods","pmids":["27486190"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"Three uncharacterized dense granule proteins identified as top fitness determinants in IFNγ-stimulated human fibroblasts share homology with GRA32, localize to dense granules, and co-immunoprecipitate with each other and with GRA32, suggesting GRA32 participates in a multi-subunit dense granule complex. Deletion of individual complex members causes early parasite egress in IFNγ-stimulated cells, implicating this GRA32-containing complex in suppression of premature egress.","method":"Genome-wide CRISPR loss-of-function screen, gene knockout, co-immunoprecipitation, dense granule localization, IFNγ-stimulated human fibroblast fitness assay","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with localization confirmation and functional KO phenotype, single lab with multiple orthogonal methods","pmids":["36916910"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"GRA32 is a dense granule protein secreted into the Toxoplasma parasitophorous vacuole that forms a multi-subunit complex with at least three paralogous dense granule proteins; this complex suppresses premature parasite egress in IFNγ-stimulated human cells, thereby promoting parasite fitness during the human immune response."},"narrative":{"mechanistic_narrative":"GRA32 is a dense granule protein of Toxoplasma gondii that is secreted into and localizes to the parasitophorous vacuole, where it functions within a multi-subunit complex to modulate parasite egress during the host immune response [PMID:27486190, PMID:36916910]. GRA32 co-immunoprecipitates with at least three paralogous, homologous dense granule proteins that are themselves top fitness determinants in IFNγ-stimulated human fibroblasts, defining a GRA32-containing complex [PMID:36916910]. Deletion of individual complex members triggers premature parasite egress in IFNγ-stimulated cells, establishing that this complex acts to suppress early egress and thereby promotes parasite fitness under immune pressure [PMID:36916910]. Beyond its complex membership and role in egress suppression, no further molecular mechanism for GRA32 has been characterized in the available corpus.","teleology":[{"year":2016,"claim":"Establishing where GRA32 acts was the first step: it was defined as a dense granule protein delivered to the parasitophorous vacuole, placing it at the host-parasite interface.","evidence":"In vivo BirA*-based proximity biotinylation with affinity purification/MS and endogenous gene tagging in Toxoplasma gondii","pmids":["27486190"],"confidence":"Medium","gaps":["No molecular activity or binding partners identified at this stage","No functional phenotype tested","Strain not specified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"The functional role and interaction context of GRA32 were resolved by showing it belongs to a multi-subunit dense granule complex whose members suppress premature parasite egress specifically in IFNγ-activated human cells.","evidence":"Genome-wide CRISPR loss-of-function screen, gene knockout, reciprocal co-immunoprecipitation, dense granule localization, and IFNγ-stimulated human fibroblast fitness assay in Toxoplasma gondii","pmids":["36916910"],"confidence":"Medium","gaps":["The molecular mechanism by which the complex suppresses egress is not defined","The direct host or parasite targets of the complex are unknown","Stoichiometry and structural organization of the complex are not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How the GRA32-containing complex mechanistically restrains egress under IFNγ stimulation, and what host or parasite pathways it engages, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No enzymatic or biochemical activity assigned to GRA32","No identified downstream effector or host target","No structural model of the complex"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["GRA32-containing multi-subunit dense granule complex (GRA32 plus three homologous dense granule proteins)"],"partners":["THREE PARALOGOUS/HOMOLOGOUS DENSE GRANULE PROTEINS THAT CO-IMMUNOPRECIPITATE WITH GRA32 [#1]"],"other_free_text":["Secreted dense granule protein localized to the Toxoplasma parasitophorous vacuole [#0]","Complex members are top fitness determinants in IFNγ-stimulated human fibroblasts; deletion causes premature parasite egress [#1]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"27486190","id":"PMC_27486190","title":"In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.","date":"2016","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/27486190","citation_count":117,"is_preprint":false},{"pmid":"36916910","id":"PMC_36916910","title":"CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/36916910","citation_count":29,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":3815,"output_tokens":653,"usd":0.01062,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5368,"output_tokens":1437,"usd":0.031383,"stage2_stop_reason":"end_turn"},"total_usd":0.042003,"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\": 2016,\n      \"finding\": \"GRA32 was identified as a dense granule protein localized to the Toxoplasma parasitophorous vacuole, detected by in vivo BirA*-based proximity biotinylation and affinity purification/mass spectrometry, with localization confirmed by endogenous gene tagging.\",\n      \"method\": \"In vivo proximity biotinylation (BirA* fusion), affinity purification, mass spectrometry, endogenous gene tagging/localization\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization confirmed by endogenous tagging plus MS identification; single lab, two orthogonal methods\",\n      \"pmids\": [\"27486190\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Three uncharacterized dense granule proteins identified as top fitness determinants in IFNγ-stimulated human fibroblasts share homology with GRA32, localize to dense granules, and co-immunoprecipitate with each other and with GRA32, suggesting GRA32 participates in a multi-subunit dense granule complex. Deletion of individual complex members causes early parasite egress in IFNγ-stimulated cells, implicating this GRA32-containing complex in suppression of premature egress.\",\n      \"method\": \"Genome-wide CRISPR loss-of-function screen, gene knockout, co-immunoprecipitation, dense granule localization, IFNγ-stimulated human fibroblast fitness assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with localization confirmation and functional KO phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36916910\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"GRA32 is a dense granule protein secreted into the Toxoplasma parasitophorous vacuole that forms a multi-subunit complex with at least three paralogous dense granule proteins; this complex suppresses premature parasite egress in IFNγ-stimulated human cells, thereby promoting parasite fitness during the human immune response.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GRA32 is a dense granule protein of Toxoplasma gondii that is secreted into and localizes to the parasitophorous vacuole, where it functions within a multi-subunit complex to modulate parasite egress during the host immune response [#0, #1]. GRA32 co-immunoprecipitates with at least three paralogous, homologous dense granule proteins that are themselves top fitness determinants in IFNγ-stimulated human fibroblasts, defining a GRA32-containing complex [#1]. Deletion of individual complex members triggers premature parasite egress in IFNγ-stimulated cells, establishing that this complex acts to suppress early egress and thereby promotes parasite fitness under immune pressure [#1]. Beyond its complex membership and role in egress suppression, no further molecular mechanism for GRA32 has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Establishing where GRA32 acts was the first step: it was defined as a dense granule protein delivered to the parasitophorous vacuole, placing it at the host-parasite interface.\",\n      \"evidence\": \"In vivo BirA*-based proximity biotinylation with affinity purification/MS and endogenous gene tagging in Toxoplasma gondii\",\n      \"pmids\": [\"27486190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No molecular activity or binding partners identified at this stage\",\n        \"No functional phenotype tested\",\n        \"Strain not specified\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The functional role and interaction context of GRA32 were resolved by showing it belongs to a multi-subunit dense granule complex whose members suppress premature parasite egress specifically in IFNγ-activated human cells.\",\n      \"evidence\": \"Genome-wide CRISPR loss-of-function screen, gene knockout, reciprocal co-immunoprecipitation, dense granule localization, and IFNγ-stimulated human fibroblast fitness assay in Toxoplasma gondii\",\n      \"pmids\": [\"36916910\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The molecular mechanism by which the complex suppresses egress is not defined\",\n        \"The direct host or parasite targets of the complex are unknown\",\n        \"Stoichiometry and structural organization of the complex are not resolved\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the GRA32-containing complex mechanistically restrains egress under IFNγ stimulation, and what host or parasite pathways it engages, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No enzymatic or biochemical activity assigned to GRA32\",\n        \"No identified downstream effector or host target\",\n        \"No structural model of the complex\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [],\n    \"complexes\": [\n      \"GRA32-containing multi-subunit dense granule complex (GRA32 plus three homologous dense granule proteins)\"\n    ],\n    \"partners\": [\n      \"Three paralogous/homologous dense granule proteins that co-immunoprecipitate with GRA32 [#1]\"\n    ],\n    \"other_free_text\": [\n      \"Secreted dense granule protein localized to the Toxoplasma parasitophorous vacuole [#0]\",\n      \"Complex members are top fitness determinants in IFNγ-stimulated human fibroblasts; deletion causes premature parasite egress [#1]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_212300","name":"GRA32","product":"dense granule protein GRA32","uniprot_acc":"S8G5E3","chromosome":"TGME49_chrX","start_pos":3931282},{"strain":"GT1","gene_id":"TGGT1_212300","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":3837742},{"strain":"RH88","gene_id":"TGRH88_043060","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023091","start_pos":3613042},{"strain":"VEG","gene_id":"TGVEG_212300","name":null,"product":"putative transmembrane protein","uniprot_acc":"B9PWK6","chromosome":"TGVEG_chrX","start_pos":3842599}]}