{"gene":"TG_236870","gene_symbol":"GRA42","run_date":"2026-09-08T15:31:32+00:00","timeline":{"discoveries":[{"year":2019,"finding":"GRA42 is a parasitophorous vacuole (PV)-localized dense granule protein individually required for induction of Lewis rat macrophage pyroptosis; deletion of GRA42 greatly reduces macrophage cell death and enhances parasite replication.","method":"Chemical mutagenesis screen, whole-genome sequencing, targeted gene deletion (Δgra42), cell death assays in Lewis rat macrophages","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with defined pyroptosis phenotype, replicated across multiple deletion combinations (single, double, triple KO), and independently confirmed by CRISPR screen in a subsequent paper","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GRA42 is required for correct localization of GRA35 (and other GRAs) to the parasitophorous vacuole membrane (PVM); in Δgra42 parasites, GRA35 and other GRAs are retained inside the parasitophorous vacuole rather than trafficking to the PVM.","method":"Immunofluorescence assay (IFA) comparing GRA35 localization in wild-type versus Δgra42 parasites","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment with functional consequence (loss of PVM targeting), supported by epistasis (double/triple KO phenotype equivalent), consistent with follow-up CRISPR screen paper","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GRA35, GRA42, and GRA43 function in the same pathway to induce Lewis rat macrophage pyroptosis, as single, double, and triple deletions of these GRAs yield similar levels of macrophage viability.","method":"Genetic epistasis analysis using single, double, and triple gene deletion parasites with cell viability readouts","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — formal epistasis demonstrated by comparing single, double, and triple KO phenotypes with quantitative cell death assay; replicated by CRISPR screen in subsequent study","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GRA42, GRA43, and GRA35 are required for parasite in vivo fitness in F344 rats (a Toxoplasma-susceptible strain), independently of their role in macrophage pyroptosis, as Δgra35, Δgra42, and Δgra43 parasites had reduced brain cyst numbers in F344 rats.","method":"In vivo rat infection model with Δgra42 and other knockout parasites; brain cyst quantification in F344 rats","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO phenotype with clear readout (cyst numbers), single lab, no orthogonal mechanism identified for the pyroptosis-independent fitness role","pmids":["30622189"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"Genome-wide CRISPR screen independently confirmed GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death (pyroptosis) in Lewis rat macrophages.","method":"Genome-wide CRISPR screen in Lewis rat macrophages infected with Toxoplasma gondii","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased genome-wide CRISPR screen replicates the earlier mutagenesis finding for GRA42's requirement in pyroptosis induction","pmids":["38376248"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"GRA42 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole and is required, together with GRA35 and GRA43 in the same pathway, for the correct trafficking of GRA35 (and other GRAs) to the parasitophorous vacuole membrane and for induction of NLRP1 inflammasome-dependent pyroptosis in Lewis rat macrophages; it is also required for parasite in vivo fitness in susceptible rat hosts independently of its role in pyroptosis."},"narrative":{"mechanistic_narrative":"GRA42 is a Toxoplasma gondii parasitophorous vacuole (PV)-localized dense granule protein that acts as an effector controlling the correct trafficking of other GRA proteins and the activation of host cell death [PMID:30622189]. GRA42 is required for the proper localization of GRA35 and additional GRAs to the parasitophorous vacuole membrane (PVM); in Δgra42 parasites these GRAs are retained inside the PV rather than reaching the PVM [PMID:30622189]. GRA42 functions in a common pathway with GRA35 and GRA43, since single, double, and triple deletions of these genes produce equivalent loss of Lewis rat macrophage pyroptosis [PMID:30622189], and deletion of GRA42 greatly reduces macrophage death while enhancing parasite replication [PMID:30622189]; an unbiased genome-wide CRISPR screen independently confirmed GRA35, GRA42, and GRA43 as the effectors mediating this cell death [PMID:38376248]. Beyond its role in pyroptosis, GRA42 is also required for parasite in vivo fitness in susceptible F344 rats, as Δgra42 parasites form fewer brain cysts, a role independent of macrophage pyroptosis [PMID:30622189]. The molecular activity of GRA42 and the mechanism by which it directs GRA trafficking have not been characterized in the available corpus.","teleology":[{"year":2019,"claim":"Establishing that a defined parasite effector controls host macrophage cell death, GRA42 was identified as a PV-localized dense granule protein individually required for Lewis rat macrophage pyroptosis.","evidence":"Chemical mutagenesis screen with whole-genome sequencing and targeted Δgra42 deletion, assayed by macrophage cell death and parasite replication","pmids":["30622189"],"confidence":"High","gaps":["molecular activity of GRA42 not defined","direct host target linking GRA42 to pyroptosis not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"To explain how GRA42 acts, it was shown to be required for trafficking of GRA35 and other GRAs from the PV lumen to the PVM, positioning GRA42 as a mediator of effector localization rather than a direct death inducer.","evidence":"Immunofluorescence comparison of GRA35 localization in wild-type versus Δgra42 parasites","pmids":["30622189"],"confidence":"High","gaps":["mechanism by which GRA42 directs GRA trafficking to the PVM unknown","whether GRA42 physically interacts with the trafficked GRAs not shown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Genetic epistasis placed GRA35, GRA42, and GRA43 in a single pathway for pyroptosis induction, defining their functional relationship.","evidence":"Comparison of single, double, and triple deletion parasites with quantitative macrophage viability readouts","pmids":["30622189"],"confidence":"High","gaps":["molecular hierarchy among the three GRAs within the pathway not resolved","host inflammasome components engaged not defined in this evidence"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"A distinct in vivo role emerged when Δgra42 (and Δgra35, Δgra43) parasites showed reduced brain cyst burden in susceptible F344 rats, establishing a pyroptosis-independent fitness function.","evidence":"In vivo F344 rat infection with knockout parasites and brain cyst quantification","pmids":["30622189"],"confidence":"Medium","gaps":["no orthogonal mechanism identified for the pyroptosis-independent fitness role","single-lab in vivo finding"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"An unbiased genome-wide screen validated the earlier candidate-based findings, confirming GRA35, GRA42, and GRA43 as the parasite effectors driving macrophage pyroptosis.","evidence":"Genome-wide CRISPR screen in Lewis rat macrophages infected with Toxoplasma gondii","pmids":["38376248"],"confidence":"High","gaps":["screen confirms requirement but does not define GRA42 biochemical function","does not resolve the trafficking mechanism"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The molecular activity of GRA42 and the mechanism by which it directs GRA trafficking to the PVM and enables NLRP1-dependent pyroptosis remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["no biochemical activity assigned to GRA42","no structural model or interaction partner identified","mechanistic basis of the pyroptosis-independent in vivo fitness role unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["GRA35 (SAME PYROPTOSIS PATHWAY; TRAFFICKING DEPENDENT ON GRA42)","GRA43 (SAME PYROPTOSIS PATHWAY)"],"other_free_text":["GRA42 is required for correct trafficking of GRA35 and other GRAs from the PV lumen to the parasitophorous vacuole membrane.","Deletion of GRA42 enhances parasite replication in Lewis rat macrophages by preventing host cell pyroptosis.","GRA42 contributes to parasite in vivo fitness (brain cyst formation) in susceptible F344 rats independently of its pyroptosis role."]},"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":"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":5270,"output_tokens":1341,"usd":0.017962,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6412,"output_tokens":2556,"usd":0.04798,"stage2_stop_reason":"end_turn"},"total_usd":0.0,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard","skipped":true},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA42 is a parasitophorous vacuole (PV)-localized dense granule protein individually required for induction of Lewis rat macrophage pyroptosis; deletion of GRA42 greatly reduces macrophage cell death and enhances parasite replication.\",\n      \"method\": \"Chemical mutagenesis screen, whole-genome sequencing, targeted gene deletion (Δgra42), cell death assays in Lewis rat macrophages\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with defined pyroptosis phenotype, replicated across multiple deletion combinations (single, double, triple KO), and independently confirmed by CRISPR screen in a subsequent paper\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA42 is required for correct localization of GRA35 (and other GRAs) to the parasitophorous vacuole membrane (PVM); in Δgra42 parasites, GRA35 and other GRAs are retained inside the parasitophorous vacuole rather than trafficking to the PVM.\",\n      \"method\": \"Immunofluorescence assay (IFA) comparing GRA35 localization in wild-type versus Δgra42 parasites\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment with functional consequence (loss of PVM targeting), supported by epistasis (double/triple KO phenotype equivalent), consistent with follow-up CRISPR screen paper\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA35, GRA42, and GRA43 function in the same pathway to induce Lewis rat macrophage pyroptosis, as single, double, and triple deletions of these GRAs yield similar levels of macrophage viability.\",\n      \"method\": \"Genetic epistasis analysis using single, double, and triple gene deletion parasites with cell viability readouts\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — formal epistasis demonstrated by comparing single, double, and triple KO phenotypes with quantitative cell death assay; replicated by CRISPR screen in subsequent study\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA42, GRA43, and GRA35 are required for parasite in vivo fitness in F344 rats (a Toxoplasma-susceptible strain), independently of their role in macrophage pyroptosis, as Δgra35, Δgra42, and Δgra43 parasites had reduced brain cyst numbers in F344 rats.\",\n      \"method\": \"In vivo rat infection model with Δgra42 and other knockout parasites; brain cyst quantification in F344 rats\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO phenotype with clear readout (cyst numbers), single lab, no orthogonal mechanism identified for the pyroptosis-independent fitness role\",\n      \"pmids\": [\"30622189\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Genome-wide CRISPR screen independently confirmed GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death (pyroptosis) in Lewis rat macrophages.\",\n      \"method\": \"Genome-wide CRISPR screen in Lewis rat macrophages infected with Toxoplasma gondii\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased genome-wide CRISPR screen replicates the earlier mutagenesis finding for GRA42's requirement in pyroptosis induction\",\n      \"pmids\": [\"38376248\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"GRA42 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole and is required, together with GRA35 and GRA43 in the same pathway, for the correct trafficking of GRA35 (and other GRAs) to the parasitophorous vacuole membrane and for induction of NLRP1 inflammasome-dependent pyroptosis in Lewis rat macrophages; it is also required for parasite in vivo fitness in susceptible rat hosts independently of its role in pyroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA42 is a Toxoplasma gondii parasitophorous vacuole (PV)-localized dense granule protein that acts as an effector controlling the correct trafficking of other GRA proteins and the activation of host cell death [#0, #1]. GRA42 is required for the proper localization of GRA35 and additional GRAs to the parasitophorous vacuole membrane (PVM); in \\u0394gra42 parasites these GRAs are retained inside the PV rather than reaching the PVM [#1]. GRA42 functions in a common pathway with GRA35 and GRA43, since single, double, and triple deletions of these genes produce equivalent loss of Lewis rat macrophage pyroptosis [#2], and deletion of GRA42 greatly reduces macrophage death while enhancing parasite replication [#0]; an unbiased genome-wide CRISPR screen independently confirmed GRA35, GRA42, and GRA43 as the effectors mediating this cell death [#4]. Beyond its role in pyroptosis, GRA42 is also required for parasite in vivo fitness in susceptible F344 rats, as \\u0394gra42 parasites form fewer brain cysts, a role independent of macrophage pyroptosis [#3]. The molecular activity of GRA42 and the mechanism by which it directs GRA trafficking have not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Establishing that a defined parasite effector controls host macrophage cell death, GRA42 was identified as a PV-localized dense granule protein individually required for Lewis rat macrophage pyroptosis.\",\n      \"evidence\": \"Chemical mutagenesis screen with whole-genome sequencing and targeted \\u0394gra42 deletion, assayed by macrophage cell death and parasite replication\",\n      \"pmids\": [\n        \"30622189\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"molecular activity of GRA42 not defined\",\n        \"direct host target linking GRA42 to pyroptosis not identified\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"To explain how GRA42 acts, it was shown to be required for trafficking of GRA35 and other GRAs from the PV lumen to the PVM, positioning GRA42 as a mediator of effector localization rather than a direct death inducer.\",\n      \"evidence\": \"Immunofluorescence comparison of GRA35 localization in wild-type versus \\u0394gra42 parasites\",\n      \"pmids\": [\n        \"30622189\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"mechanism by which GRA42 directs GRA trafficking to the PVM unknown\",\n        \"whether GRA42 physically interacts with the trafficked GRAs not shown\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic epistasis placed GRA35, GRA42, and GRA43 in a single pathway for pyroptosis induction, defining their functional relationship.\",\n      \"evidence\": \"Comparison of single, double, and triple deletion parasites with quantitative macrophage viability readouts\",\n      \"pmids\": [\n        \"30622189\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"molecular hierarchy among the three GRAs within the pathway not resolved\",\n        \"host inflammasome components engaged not defined in this evidence\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A distinct in vivo role emerged when \\u0394gra42 (and \\u0394gra35, \\u0394gra43) parasites showed reduced brain cyst burden in susceptible F344 rats, establishing a pyroptosis-independent fitness function.\",\n      \"evidence\": \"In vivo F344 rat infection with knockout parasites and brain cyst quantification\",\n      \"pmids\": [\n        \"30622189\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"no orthogonal mechanism identified for the pyroptosis-independent fitness role\",\n        \"single-lab in vivo finding\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"An unbiased genome-wide screen validated the earlier candidate-based findings, confirming GRA35, GRA42, and GRA43 as the parasite effectors driving macrophage pyroptosis.\",\n      \"evidence\": \"Genome-wide CRISPR screen in Lewis rat macrophages infected with Toxoplasma gondii\",\n      \"pmids\": [\n        \"38376248\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"screen confirms requirement but does not define GRA42 biochemical function\",\n        \"does not resolve the trafficking mechanism\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular activity of GRA42 and the mechanism by which it directs GRA trafficking to the PVM and enables NLRP1-dependent pyroptosis remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"no biochemical activity assigned to GRA42\",\n        \"no structural model or interaction partner identified\",\n        \"mechanistic basis of the pyroptosis-independent in vivo fitness role unknown\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0020003\",\n        \"supporting_discovery_ids\": [0, 1],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"GO:0070269\",\n        \"supporting_discovery_ids\": [0, 2, 4],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"GRA35 (same pyroptosis pathway; trafficking dependent on GRA42)\",\n      \"GRA43 (same pyroptosis pathway)\"\n    ],\n    \"other_free_text\": [\n      \"GRA42 is required for correct trafficking of GRA35 and other GRAs from the PV lumen to the parasitophorous vacuole membrane.\",\n      \"Deletion of GRA42 enhances parasite replication in Lewis rat macrophages by preventing host cell pyroptosis.\",\n      \"GRA42 contributes to parasite in vivo fitness (brain cyst formation) in susceptible F344 rats independently of its pyroptosis role.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_236870","name":"GRA42","product":"dense granule protein GRA42","uniprot_acc":"S8EW90","chromosome":"TGME49_chrX","start_pos":5561579},{"strain":"GT1","gene_id":"TGGT1_236870","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":5454717},{"strain":"RH88","gene_id":"TGRH88_041070","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023091","start_pos":1972326},{"strain":"VEG","gene_id":"TGVEG_236870","name":null,"product":"putative transmembrane protein","uniprot_acc":"B9Q6J4","chromosome":"TGVEG_chrX","start_pos":5488067}]}