{"gene":"TG_204270","gene_symbol":"GRA60","run_date":"2026-09-08T11:06:14","timeline":{"discoveries":[{"year":2020,"finding":"GRA60 is a dense granule protein that localizes to the vacuolar space and parasitophorous vacuolar membrane (PVM) in Toxoplasma gondii type I RH strain.","method":"Direct localization experiment (microscopy/fractionation of dense granule protein)","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional context in a single lab study using multiple observations","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"GRA60 contributes to virulence in mice; deletion of GRA60 from the RH strain results in reduced virulence, establishing a functional role in pathogenesis.","method":"Loss-of-function (gene knockout) with in vivo virulence assay in mice","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined in vivo phenotype, single lab, single study","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"Deletion of GRA60 in the RH strain leads to recruitment of host IRG family members Irgb10 and Irga6 to the PVM, demonstrating that GRA60 normally inhibits IRG-mediated host defense at the PVM.","method":"Loss-of-function (gene knockout) with immunofluorescence microscopy monitoring IRG recruitment to the PVM","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with specific molecular readout (IRG recruitment), supported by comparison to known virulence mutants in the same study","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"Epistasis analysis shows that deletion of GRA60 in an RHΔrop5 background results in IRG recruitment comparable to either single knockout alone, indicating GRA60 and ROP5 operate in the same pathway/complex for IRG resistance rather than additively.","method":"Genetic epistasis (double-knockout construction RHΔgra60Δrop5 vs. single knockouts, with IRG recruitment assay)","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via double-mutant analysis with specific molecular phenotype readout, single lab","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"GRA60 is found physically associated with ROP18, a serine-threonine kinase component of the virulence complex, suggesting GRA60 functions as part of the ROP18-containing effector complex that inhibits IRG-mediated host immunity.","method":"Co-immunoprecipitation / association assay (GRA60 found associated with ROP18)","journal":"Cellular microbiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single association reported in abstract without reciprocal validation details; single lab, single study","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"GRA60 is a Toxoplasma gondii dense granule effector protein that localizes to the vacuolar space and PVM, where it associates with the virulence kinase ROP18 and functions—in concert with ROP5 and ROP18—to inhibit the recruitment of host immunity-related GTPases (Irgb10 and Irga6) to the PVM, thereby protecting the parasite from host cell-autonomous immune destruction and contributing to virulence in mice."},"narrative":{"mechanistic_narrative":"GRA60 is a Toxoplasma gondii dense granule effector that protects the intracellular parasite from host cell-autonomous immunity [PMID:33040458]. In the type I RH strain, GRA60 localizes to the vacuolar space and the parasitophorous vacuolar membrane (PVM) [PMID:33040458], where it functions to block recruitment of host immunity-related GTPases Irgb10 and Irga6 to the PVM: deletion of GRA60 permits IRG loading onto the vacuole [PMID:33040458] and reduces parasite virulence in mice [PMID:33040458]. Epistasis analysis places GRA60 in the same IRG-resistance pathway as ROP5 rather than acting additively [PMID:33040458], and GRA60 physically associates with the virulence kinase ROP18 [PMID:33040458], indicating it operates within the ROP5/ROP18 virulence-effector machinery at the PVM. Beyond this genetic and localization framework in the RH strain, the biochemical mechanism by which GRA60 acts within the complex has not been characterized in the available corpus.","teleology":[{"year":2020,"claim":"Establishing where GRA60 resides was the first step in assigning it a role; localization to the vacuolar space and PVM placed it at the host-parasite interface where immune effectors are engaged.","evidence":"Direct localization of the dense granule protein by microscopy/fractionation in RH strain","pmids":["33040458"],"confidence":"Medium","gaps":["Does not define how GRA60 is trafficked to or retained at the PVM","No topology or membrane-association mechanism established"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"Whether GRA60 mattered for pathogenesis was tested by knockout, showing it is required for full virulence and thereby linking the protein to a functional in vivo role.","evidence":"Gene knockout in RH strain with in vivo mouse virulence assay","pmids":["33040458"],"confidence":"Medium","gaps":["Does not distinguish direct immune-evasion contribution from other pathways","Single strain background tested"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"The molecular basis of the virulence defect was pinpointed by showing GRA60 loss permits Irgb10 and Irga6 recruitment to the PVM, establishing GRA60 as an inhibitor of IRG-mediated host defense.","evidence":"Knockout combined with immunofluorescence monitoring IRG recruitment to the PVM in RH strain","pmids":["33040458"],"confidence":"Medium","gaps":["Mechanism of IRG exclusion (direct blocking vs. modification) unknown","Does not address other IRG or GBP family members"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"To locate GRA60 within known virulence machinery, epistasis and association were tested, placing it in the same pathway as ROP5 and physically linking it to the ROP18 kinase.","evidence":"Double-knockout epistasis (RHΔgra60Δrop5) with IRG readout, plus Co-IP association of GRA60 with ROP18","pmids":["33040458"],"confidence":"Low","gaps":["ROP18 association is a single Co-IP without reciprocal validation","Whether GRA60 is a ROP18 substrate, regulator, or scaffold is undetermined","Stoichiometry and architecture of the GRA60-ROP5-ROP18 complex unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":null,"claim":"The biochemical function of GRA60 within the ROP5/ROP18 complex and whether its role is conserved beyond the type I RH strain remain open.","evidence":"No further mechanistic characterization in the available corpus","pmids":[],"confidence":"Low","gaps":["No structural or enzymatic activity defined for GRA60","No characterization in type II or III strains","Direct molecular interaction of GRA60 with IRGs untested"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["ROP5/ROP18-containing virulence effector complex"],"partners":["ROP18","ROP5 (SAME GENETIC PATHWAY)"],"other_free_text":["GRA60 deletion permits recruitment of host IRGs Irgb10 and Irga6 to the PVM","GRA60 is required for full virulence in mice in the RH strain","Epistasis places GRA60 and ROP5 in a single IRG-resistance pathway rather than additive pathways"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"33040458","id":"PMC_33040458","title":"Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence.","date":"2020","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/33040458","citation_count":24,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":2926,"output_tokens":1184,"usd":0.013269,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6169,"output_tokens":2173,"usd":0.042585,"stage2_stop_reason":"end_turn"},"total_usd":0.055854,"stage1_batch_id":"msgbatch_01WokvBYTB6JLnYrpD4rXnyV","stage2_batch_id":"msgbatch_01M7hsQDvz6AqVC9DLVjQzzc","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA60 is a dense granule protein that localizes to the vacuolar space and parasitophorous vacuolar membrane (PVM) in Toxoplasma gondii type I RH strain.\",\n      \"method\": \"Direct localization experiment (microscopy/fractionation of dense granule protein)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional context in a single lab study using multiple observations\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA60 contributes to virulence in mice; deletion of GRA60 from the RH strain results in reduced virulence, establishing a functional role in pathogenesis.\",\n      \"method\": \"Loss-of-function (gene knockout) with in vivo virulence assay in mice\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined in vivo phenotype, single lab, single study\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Deletion of GRA60 in the RH strain leads to recruitment of host IRG family members Irgb10 and Irga6 to the PVM, demonstrating that GRA60 normally inhibits IRG-mediated host defense at the PVM.\",\n      \"method\": \"Loss-of-function (gene knockout) with immunofluorescence microscopy monitoring IRG recruitment to the PVM\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with specific molecular readout (IRG recruitment), supported by comparison to known virulence mutants in the same study\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Epistasis analysis shows that deletion of GRA60 in an RHΔrop5 background results in IRG recruitment comparable to either single knockout alone, indicating GRA60 and ROP5 operate in the same pathway/complex for IRG resistance rather than additively.\",\n      \"method\": \"Genetic epistasis (double-knockout construction RHΔgra60Δrop5 vs. single knockouts, with IRG recruitment assay)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via double-mutant analysis with specific molecular phenotype readout, single lab\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA60 is found physically associated with ROP18, a serine-threonine kinase component of the virulence complex, suggesting GRA60 functions as part of the ROP18-containing effector complex that inhibits IRG-mediated host immunity.\",\n      \"method\": \"Co-immunoprecipitation / association assay (GRA60 found associated with ROP18)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single association reported in abstract without reciprocal validation details; single lab, single study\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"GRA60 is a Toxoplasma gondii dense granule effector protein that localizes to the vacuolar space and PVM, where it associates with the virulence kinase ROP18 and functions—in concert with ROP5 and ROP18—to inhibit the recruitment of host immunity-related GTPases (Irgb10 and Irga6) to the PVM, thereby protecting the parasite from host cell-autonomous immune destruction and contributing to virulence in mice.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA60 is a Toxoplasma gondii dense granule effector that protects the intracellular parasite from host cell-autonomous immunity [#0, #2]. In the type I RH strain, GRA60 localizes to the vacuolar space and the parasitophorous vacuolar membrane (PVM) [#0], where it functions to block recruitment of host immunity-related GTPases Irgb10 and Irga6 to the PVM: deletion of GRA60 permits IRG loading onto the vacuole [#2] and reduces parasite virulence in mice [#1]. Epistasis analysis places GRA60 in the same IRG-resistance pathway as ROP5 rather than acting additively [#3], and GRA60 physically associates with the virulence kinase ROP18 [#4], indicating it operates within the ROP5/ROP18 virulence-effector machinery at the PVM. Beyond this genetic and localization framework in the RH strain, the biochemical mechanism by which GRA60 acts within the complex has not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing where GRA60 resides was the first step in assigning it a role; localization to the vacuolar space and PVM placed it at the host-parasite interface where immune effectors are engaged.\",\n      \"evidence\": \"Direct localization of the dense granule protein by microscopy/fractionation in RH strain\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Does not define how GRA60 is trafficked to or retained at the PVM\",\n        \"No topology or membrane-association mechanism established\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether GRA60 mattered for pathogenesis was tested by knockout, showing it is required for full virulence and thereby linking the protein to a functional in vivo role.\",\n      \"evidence\": \"Gene knockout in RH strain with in vivo mouse virulence assay\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Does not distinguish direct immune-evasion contribution from other pathways\",\n        \"Single strain background tested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The molecular basis of the virulence defect was pinpointed by showing GRA60 loss permits Irgb10 and Irga6 recruitment to the PVM, establishing GRA60 as an inhibitor of IRG-mediated host defense.\",\n      \"evidence\": \"Knockout combined with immunofluorescence monitoring IRG recruitment to the PVM in RH strain\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of IRG exclusion (direct blocking vs. modification) unknown\",\n        \"Does not address other IRG or GBP family members\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"To locate GRA60 within known virulence machinery, epistasis and association were tested, placing it in the same pathway as ROP5 and physically linking it to the ROP18 kinase.\",\n      \"evidence\": \"Double-knockout epistasis (RH\\u0394gra60\\u0394rop5) with IRG readout, plus Co-IP association of GRA60 with ROP18\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"ROP18 association is a single Co-IP without reciprocal validation\",\n        \"Whether GRA60 is a ROP18 substrate, regulator, or scaffold is undetermined\",\n        \"Stoichiometry and architecture of the GRA60-ROP5-ROP18 complex unresolved\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The biochemical function of GRA60 within the ROP5/ROP18 complex and whether its role is conserved beyond the type I RH strain remain open.\",\n      \"evidence\": \"No further mechanistic characterization in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural or enzymatic activity defined for GRA60\",\n        \"No characterization in type II or III strains\",\n        \"Direct molecular interaction of GRA60 with IRGs untested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0052150\", \"supporting_discovery_ids\": [2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051702\", \"supporting_discovery_ids\": [1, 2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [\n      \"ROP5/ROP18-containing virulence effector complex\"\n    ],\n    \"partners\": [\n      \"ROP18\",\n      \"ROP5 (same genetic pathway)\"\n    ],\n    \"other_free_text\": [\n      \"GRA60 deletion permits recruitment of host IRGs Irgb10 and Irga6 to the PVM\",\n      \"GRA60 is required for full virulence in mice in the RH strain\",\n      \"Epistasis places GRA60 and ROP5 in a single IRG-resistance pathway rather than additive pathways\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_204270","name":"GRA60","product":"dense granule protein GRA60","uniprot_acc":"S8GNA6","chromosome":"TGME49_chrVIIa","start_pos":1931594},{"strain":"GT1","gene_id":"TGGT1_204270","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrVIIa","start_pos":1833641},{"strain":"RH88","gene_id":"TGRH88_034870","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023088","start_pos":2023255},{"strain":"VEG","gene_id":"TGVEG_204270","name":null,"product":"putative transmembrane protein","uniprot_acc":"B9QFF7","chromosome":"TGVEG_chrVIIa","start_pos":2015222}]}