{"gene":"TG_231960","gene_symbol":"GRA28","run_date":"2026-09-08T12:58:24","timeline":{"discoveries":[{"year":2022,"finding":"GRA28 is secreted into the host cell nucleus via the MYR1 secretory/translocon pathway, where it cooperates with host chromatin remodelers to drive CCR7 upregulation and chemotactic migration of parasitized macrophages, conferring dendritic cell-like migratory properties.","method":"Genetic knockout of GRA28 and MYR1 with adoptive transfer in vivo migration assays, nuclear localization confirmed, chromatin remodeler cooperation established","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic knockouts (ΔGRA28, ΔMYR1), in vivo competitive adoptive transfer, nuclear localization, replicated across multiple labs","pmids":["36309013"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"GRA28 is secreted into the host cell nucleus and is required for T. gondii-induced CCL22 chemokine production in human placental cells, human monocyte cell lines, and mouse immune and placental cells both in vitro and in vivo; deletion of GRA28 impairs parasite dissemination in vivo.","method":"CRISPR-based GRA28 deletion, nuclear localization by imaging, CCL22 ELISA in human and mouse cells, in vivo dissemination assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with defined molecular phenotype (CCL22 reduction), nuclear localization confirmed, in vitro and in vivo readouts, human and mouse cells","pmids":["34781732"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"TgPPM3C, a secreted PP2C-class phosphatase, is required for GRA28 export into the host cell; phosphoproteomic analysis showed altered phosphorylation of GRA28 in ΔTgPPM3C parasites, and GRA28 export was defective in these mutants, suggesting dephosphorylation by TgPPM3C is a critical step for GRA28 translocation.","method":"Phosphoproteomics of ΔTgPPM3C parasites, GRA16/GRA28 export assays in TgPPM3C knockout parasites","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics plus export assay in clean KO, single lab, two orthogonal methods","pmids":["33370417"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"GRA28 boosts chromatin accessibility at the Ccr7 gene locus in primary macrophages and enhances GRA15/GRA24/NF-κB-dependent transcription, thereby cooperating with GRA15 and GRA24 to promote CCR7-mediated macrophage chemotaxis and parasite dissemination.","method":"Combinatorial GRA knockout parasites, chromatin accessibility assays (ATAC-seq implied), in vivo competitive adoptive transfer of infected macrophages","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — combinatorial knockouts, chromatin accessibility assays, in vivo competitive dissemination, replicated in peer-reviewed publication matching preprint","pmids":["39207098","38370679"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2024,"finding":"GRA28 is one of four nuclear-targeted MYR1-dependent effectors (TgIST, GRA16, GRA24, GRA28) required to block IFNγ-driven host cell death and parasite premature egress in human cells; specifically, GRA28 disrupts histone acetyltransferase activity of CBP/p300 and directly influences IFNγ-driven transcription.","method":"Genome-wide loss-of-function screens in IFNγ-primed human cells, combinatorial GRA knockouts, transcription assays, CBP/p300 acetyltransferase activity assay","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen plus targeted KO with defined molecular mechanism (CBP/p300 inhibition), single lab, multiple methods","pmids":["39292011","38234811"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"GRA28 is one of eight MYR1-dependent dense granule effectors (IST, NSM, HCE1/TEEGR, GRA16, GRA18, GRA24, GRA28, GRA84) that cooperate in an additive network to activate the non-canonical NF-κB pathway (RelB/p52), acting upstream of TRAF3 depletion, NIK stabilization, and p100 processing to p52.","method":"Combinatorial knockout parasite panel, nuclear accumulation assays for RelB/p52, epistasis with MYR1 deletion","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — combinatorial KO panel with defined pathway placement, preprint not yet peer-reviewed, single lab","pmids":["41959509"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2018,"finding":"CRISPR-Cas9 deletion of GRA28 in the virulent RH strain had no significant effect on in vitro growth, egress, or virulence in BALB/c mice, indicating GRA28 is dispensable for these processes in the RH strain.","method":"CRISPR-Cas9 knockout, plaque assay, egress assay, mouse virulence model","journal":"Frontiers in cellular and infection microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with multiple in vitro and in vivo readouts; finding is a negative result (no phenotype in RH)","pmids":["30211128"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"GRA28 is a dense granule effector secreted via the MYR1 translocon into the host cell nucleus, where it cooperates with host chromatin remodelers (boosting chromatin accessibility at immune gene loci) and disrupts CBP/p300 histone acetyltransferase activity to drive CCL22 production, CCR7 upregulation, macrophage hypermigration, and blockade of IFNγ-driven host cell death, collectively promoting parasite dissemination; its export requires prior dephosphorylation by the vacuolar phosphatase TgPPM3C."},"narrative":{"mechanistic_narrative":"GRA28 is a Toxoplasma gondii dense granule effector that is secreted into the host cell nucleus via the MYR1 translocon, where it reprograms host gene expression to promote immune evasion and parasite dissemination [PMID:36309013, PMID:34781732]. Its export from the vacuole depends on prior dephosphorylation by the secreted PP2C-class phosphatase TgPPM3C, and GRA28 export is defective in ΔTgPPM3C parasites [PMID:33370417]. In the nucleus, GRA28 cooperates with host chromatin remodelers, boosting chromatin accessibility at the Ccr7 locus and enhancing GRA15/GRA24/NF-κB-dependent transcription to drive CCR7 upregulation and confer dendritic cell-like hypermigratory properties on parasitized macrophages [PMID:36309013, PMID:39207098, PMID:38370679]. GRA28 is also required for T. gondii-induced CCL22 chemokine production across human and mouse immune and placental cells, and its deletion impairs parasite dissemination in vivo [PMID:34781732]. As one of several nuclear MYR1-dependent effectors, GRA28 disrupts the histone acetyltransferase activity of CBP/p300 and influences IFNγ-driven transcription, contributing to blockade of IFNγ-driven host cell death [PMID:39292011, PMID:38234811]. Within the effector network, GRA28 acts additively with other MYR1-dependent effectors to activate the non-canonical NF-κB (RelB/p52) pathway [PMID:41959509]. In the virulent RH strain, GRA28 deletion has no effect on in vitro growth, egress, or acute virulence, indicating its dissemination and immune-modulatory roles are separable from acute lytic fitness [PMID:30211128].","teleology":[{"year":2018,"claim":"Whether GRA28 contributes to core lytic fitness was unknown; deleting it in the virulent RH strain established that GRA28 is dispensable for in vitro growth, egress, and acute virulence, redirecting attention toward non-lytic host-modulatory functions.","evidence":"CRISPR-Cas9 knockout with plaque, egress, and mouse virulence assays in RH strain","pmids":["30211128"],"confidence":"Medium","gaps":["Negative result does not address chronic infection or dissemination phenotypes","Tested only in one strain background","No molecular function assigned"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"How GRA28 reaches the host cell was unresolved; identifying TgPPM3C as a phosphatase required for GRA28 export established dephosphorylation as a regulatory checkpoint preceding translocation.","evidence":"Phosphoproteomics and GRA16/GRA28 export assays in ΔTgPPM3C knockout parasites","pmids":["33370417"],"confidence":"Medium","gaps":["Direct dephosphorylation of GRA28 by TgPPM3C not demonstrated biochemically","Which residues are critical for export is undefined","Single lab, correlative link between phosphostate and export"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"The functional output of nuclear GRA28 was unknown; showing it is required for CCL22 induction across human and mouse cells and for in vivo dissemination assigned GRA28 a chemokine-driving, dissemination-promoting role.","evidence":"CRISPR GRA28 deletion, CCL22 ELISA in human/mouse immune and placental cells, in vivo dissemination assay","pmids":["34781732"],"confidence":"High","gaps":["Molecular mechanism linking GRA28 to CCL22 transcription not resolved","Direct host DNA/chromatin targets not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"It was unclear how GRA28 alters host cell behavior; establishing MYR1-dependent nuclear delivery and cooperation with host chromatin remodelers to drive CCR7 upregulation and macrophage hypermigration defined GRA28 as a nuclear reprogramming effector conferring migratory properties.","evidence":"Reciprocal ΔGRA28/ΔMYR1 knockouts, nuclear localization imaging, in vivo competitive adoptive transfer migration assays","pmids":["36309013"],"confidence":"High","gaps":["Identity of the cooperating chromatin remodelers not fully specified","Whether GRA28 binds chromatin directly unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"The chromatin-level mechanism was undefined; showing GRA28 boosts chromatin accessibility at the Ccr7 locus and enhances GRA15/GRA24/NF-κB-dependent transcription placed GRA28 within a cooperative effector module driving CCR7-mediated chemotaxis and dissemination.","evidence":"Combinatorial GRA knockouts, chromatin accessibility assays, in vivo competitive adoptive transfer in type II strain","pmids":["39207098","38370679"],"confidence":"High","gaps":["Whether GRA28 acts directly at the locus or via recruited factors unresolved","Biochemical partners at the chromatin level not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2024,"claim":"GRA28's role in immune-driven cell death was unknown; identifying it among nuclear MYR1-dependent effectors that block IFNγ-driven host cell death, acting by disrupting CBP/p300 acetyltransferase activity and IFNγ-driven transcription, assigned it a direct enzymatic-interference mechanism.","evidence":"Genome-wide loss-of-function screens in IFNγ-primed human cells, combinatorial GRA knockouts, transcription and CBP/p300 acetyltransferase activity assays","pmids":["39292011","38234811"],"confidence":"Medium","gaps":["Direct physical interaction between GRA28 and CBP/p300 not established","Redundancy with other nuclear effectors complicates isolating GRA28's specific contribution"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"How GRA28 fits into host NF-κB signaling was incompletely mapped; placing it as one of eight MYR1-dependent effectors that additively activate the non-canonical NF-κB (RelB/p52) pathway upstream of TRAF3 depletion and NIK stabilization defined its network-level position.","evidence":"Combinatorial knockout panel, RelB/p52 nuclear accumulation assays, epistasis with MYR1 deletion in type I and type II strains (preprint)","pmids":["41959509"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","GRA28's individual contribution within the additive network not isolated","Direct molecular target upstream of TRAF3 depletion unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":null,"claim":"The direct host molecular target(s) of GRA28 and whether it binds chromatin, CBP/p300, or NF-κB components directly remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No demonstrated direct physical interaction partner for GRA28","No structural model or defined functional domains","Mechanism by which GRA28 alters chromatin accessibility is unresolved"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["MYR1 (TRANSLOCON REQUIRED FOR GRA28 HOST EXPORT)","TGPPM3C (PHOSPHATASE REQUIRED FOR GRA28 EXPORT)","CBP/P300 (HOST HISTONE ACETYLTRANSFERASE WHOSE ACTIVITY GRA28 DISRUPTS)","GRA15, GRA24 (COOPERATING EFFECTORS IN NF-ΚB-DEPENDENT TRANSCRIPTION)"],"other_free_text":["GRA28 drives CCL22 chemokine production and CCR7 upregulation, conferring dendritic cell-like hypermigration on parasitized macrophages and promoting dissemination.","GRA28 blocks IFNγ-driven host cell death as part of a nuclear MYR1-dependent effector set (with TgIST, GRA16, GRA24).","GRA28 is dispensable for in vitro growth, egress, and acute virulence in the RH strain."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"28404792","id":"PMC_28404792","title":"Toxoplasma Effectors Targeting Host Signaling and Transcription.","date":"2017","source":"Clinical microbiology reviews","url":"https://pubmed.ncbi.nlm.nih.gov/28404792","citation_count":268,"is_preprint":false},{"pmid":"36309013","id":"PMC_36309013","title":"The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages.","date":"2022","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/36309013","citation_count":42,"is_preprint":false},{"pmid":"30211128","id":"PMC_30211128","title":"Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System.","date":"2018","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30211128","citation_count":30,"is_preprint":false},{"pmid":"34781732","id":"PMC_34781732","title":"Toxoplasma gondii GRA28 Is Required for Placenta-Specific Induction of the Regulatory Chemokine CCL22 in Human and Mouse.","date":"2021","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/34781732","citation_count":27,"is_preprint":false},{"pmid":"33370417","id":"PMC_33370417","title":"Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/33370417","citation_count":20,"is_preprint":false},{"pmid":"39207098","id":"PMC_39207098","title":"Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination.","date":"2024","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/39207098","citation_count":13,"is_preprint":false},{"pmid":"39292011","id":"PMC_39292011","title":"A combination of four Toxoplasma gondii nuclear-targeted effectors protects against interferon gamma-driven human host cell death.","date":"2024","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/39292011","citation_count":8,"is_preprint":false},{"pmid":"36470783","id":"PMC_36470783","title":"Macrophage to dendritic cell transitioning induced by Toxoplasma.","date":"2022","source":"Trends in parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/36470783","citation_count":2,"is_preprint":false},{"pmid":"38234811","id":"PMC_38234811","title":"A Combination of Four Nuclear Targeted Effectors Protects Toxoplasma Against Interferon Gamma Driven Human Host Cell Death During Acute Infection.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38234811","citation_count":1,"is_preprint":false},{"pmid":"38370679","id":"PMC_38370679","title":"Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38370679","citation_count":1,"is_preprint":false},{"pmid":"41441646","id":"PMC_41441646","title":"Protective Efficacy of Two Novel DNA Vaccine Candidates Encoding TgGRA28 and TgGRA83 with an IL-28B Molecular Adjuvant Against Acute and Chronic Toxoplasmosis in Mice.","date":"2025","source":"Vaccines","url":"https://pubmed.ncbi.nlm.nih.gov/41441646","citation_count":1,"is_preprint":false},{"pmid":"41959509","id":"PMC_41959509","title":"A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41959509","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8941,"output_tokens":2045,"usd":0.028749,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7385,"output_tokens":3696,"usd":0.064662,"stage2_stop_reason":"end_turn"},"total_usd":0.093411,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"GRA28 is secreted into the host cell nucleus via the MYR1 secretory/translocon pathway, where it cooperates with host chromatin remodelers to drive CCR7 upregulation and chemotactic migration of parasitized macrophages, conferring dendritic cell-like migratory properties.\",\n      \"method\": \"Genetic knockout of GRA28 and MYR1 with adoptive transfer in vivo migration assays, nuclear localization confirmed, chromatin remodeler cooperation established\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic knockouts (ΔGRA28, ΔMYR1), in vivo competitive adoptive transfer, nuclear localization, replicated across multiple labs\",\n      \"pmids\": [\"36309013\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GRA28 is secreted into the host cell nucleus and is required for T. gondii-induced CCL22 chemokine production in human placental cells, human monocyte cell lines, and mouse immune and placental cells both in vitro and in vivo; deletion of GRA28 impairs parasite dissemination in vivo.\",\n      \"method\": \"CRISPR-based GRA28 deletion, nuclear localization by imaging, CCL22 ELISA in human and mouse cells, in vivo dissemination assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with defined molecular phenotype (CCL22 reduction), nuclear localization confirmed, in vitro and in vivo readouts, human and mouse cells\",\n      \"pmids\": [\"34781732\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TgPPM3C, a secreted PP2C-class phosphatase, is required for GRA28 export into the host cell; phosphoproteomic analysis showed altered phosphorylation of GRA28 in ΔTgPPM3C parasites, and GRA28 export was defective in these mutants, suggesting dephosphorylation by TgPPM3C is a critical step for GRA28 translocation.\",\n      \"method\": \"Phosphoproteomics of ΔTgPPM3C parasites, GRA16/GRA28 export assays in TgPPM3C knockout parasites\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics plus export assay in clean KO, single lab, two orthogonal methods\",\n      \"pmids\": [\"33370417\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GRA28 boosts chromatin accessibility at the Ccr7 gene locus in primary macrophages and enhances GRA15/GRA24/NF-κB-dependent transcription, thereby cooperating with GRA15 and GRA24 to promote CCR7-mediated macrophage chemotaxis and parasite dissemination.\",\n      \"method\": \"Combinatorial GRA knockout parasites, chromatin accessibility assays (ATAC-seq implied), in vivo competitive adoptive transfer of infected macrophages\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — combinatorial knockouts, chromatin accessibility assays, in vivo competitive dissemination, replicated in peer-reviewed publication matching preprint\",\n      \"pmids\": [\"39207098\", \"38370679\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GRA28 is one of four nuclear-targeted MYR1-dependent effectors (TgIST, GRA16, GRA24, GRA28) required to block IFNγ-driven host cell death and parasite premature egress in human cells; specifically, GRA28 disrupts histone acetyltransferase activity of CBP/p300 and directly influences IFNγ-driven transcription.\",\n      \"method\": \"Genome-wide loss-of-function screens in IFNγ-primed human cells, combinatorial GRA knockouts, transcription assays, CBP/p300 acetyltransferase activity assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen plus targeted KO with defined molecular mechanism (CBP/p300 inhibition), single lab, multiple methods\",\n      \"pmids\": [\"39292011\", \"38234811\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GRA28 is one of eight MYR1-dependent dense granule effectors (IST, NSM, HCE1/TEEGR, GRA16, GRA18, GRA24, GRA28, GRA84) that cooperate in an additive network to activate the non-canonical NF-κB pathway (RelB/p52), acting upstream of TRAF3 depletion, NIK stabilization, and p100 processing to p52.\",\n      \"method\": \"Combinatorial knockout parasite panel, nuclear accumulation assays for RelB/p52, epistasis with MYR1 deletion\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — combinatorial KO panel with defined pathway placement, preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [\"41959509\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRISPR-Cas9 deletion of GRA28 in the virulent RH strain had no significant effect on in vitro growth, egress, or virulence in BALB/c mice, indicating GRA28 is dispensable for these processes in the RH strain.\",\n      \"method\": \"CRISPR-Cas9 knockout, plaque assay, egress assay, mouse virulence model\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with multiple in vitro and in vivo readouts; finding is a negative result (no phenotype in RH)\",\n      \"pmids\": [\"30211128\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"GRA28 is a dense granule effector secreted via the MYR1 translocon into the host cell nucleus, where it cooperates with host chromatin remodelers (boosting chromatin accessibility at immune gene loci) and disrupts CBP/p300 histone acetyltransferase activity to drive CCL22 production, CCR7 upregulation, macrophage hypermigration, and blockade of IFNγ-driven host cell death, collectively promoting parasite dissemination; its export requires prior dephosphorylation by the vacuolar phosphatase TgPPM3C.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA28 is a Toxoplasma gondii dense granule effector that is secreted into the host cell nucleus via the MYR1 translocon, where it reprograms host gene expression to promote immune evasion and parasite dissemination [#0, #1]. Its export from the vacuole depends on prior dephosphorylation by the secreted PP2C-class phosphatase TgPPM3C, and GRA28 export is defective in ΔTgPPM3C parasites [#2]. In the nucleus, GRA28 cooperates with host chromatin remodelers, boosting chromatin accessibility at the Ccr7 locus and enhancing GRA15/GRA24/NF-κB-dependent transcription to drive CCR7 upregulation and confer dendritic cell-like hypermigratory properties on parasitized macrophages [#0, #3]. GRA28 is also required for T. gondii-induced CCL22 chemokine production across human and mouse immune and placental cells, and its deletion impairs parasite dissemination in vivo [#1]. As one of several nuclear MYR1-dependent effectors, GRA28 disrupts the histone acetyltransferase activity of CBP/p300 and influences IFNγ-driven transcription, contributing to blockade of IFNγ-driven host cell death [#4]. Within the effector network, GRA28 acts additively with other MYR1-dependent effectors to activate the non-canonical NF-κB (RelB/p52) pathway [#5]. In the virulent RH strain, GRA28 deletion has no effect on in vitro growth, egress, or acute virulence, indicating its dissemination and immune-modulatory roles are separable from acute lytic fitness [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Whether GRA28 contributes to core lytic fitness was unknown; deleting it in the virulent RH strain established that GRA28 is dispensable for in vitro growth, egress, and acute virulence, redirecting attention toward non-lytic host-modulatory functions.\",\n      \"evidence\": \"CRISPR-Cas9 knockout with plaque, egress, and mouse virulence assays in RH strain\",\n      \"pmids\": [\"30211128\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Negative result does not address chronic infection or dissemination phenotypes\",\n        \"Tested only in one strain background\",\n        \"No molecular function assigned\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"How GRA28 reaches the host cell was unresolved; identifying TgPPM3C as a phosphatase required for GRA28 export established dephosphorylation as a regulatory checkpoint preceding translocation.\",\n      \"evidence\": \"Phosphoproteomics and GRA16/GRA28 export assays in ΔTgPPM3C knockout parasites\",\n      \"pmids\": [\"33370417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct dephosphorylation of GRA28 by TgPPM3C not demonstrated biochemically\",\n        \"Which residues are critical for export is undefined\",\n        \"Single lab, correlative link between phosphostate and export\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The functional output of nuclear GRA28 was unknown; showing it is required for CCL22 induction across human and mouse cells and for in vivo dissemination assigned GRA28 a chemokine-driving, dissemination-promoting role.\",\n      \"evidence\": \"CRISPR GRA28 deletion, CCL22 ELISA in human/mouse immune and placental cells, in vivo dissemination assay\",\n      \"pmids\": [\"34781732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular mechanism linking GRA28 to CCL22 transcription not resolved\",\n        \"Direct host DNA/chromatin targets not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"It was unclear how GRA28 alters host cell behavior; establishing MYR1-dependent nuclear delivery and cooperation with host chromatin remodelers to drive CCR7 upregulation and macrophage hypermigration defined GRA28 as a nuclear reprogramming effector conferring migratory properties.\",\n      \"evidence\": \"Reciprocal ΔGRA28/ΔMYR1 knockouts, nuclear localization imaging, in vivo competitive adoptive transfer migration assays\",\n      \"pmids\": [\"36309013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the cooperating chromatin remodelers not fully specified\",\n        \"Whether GRA28 binds chromatin directly unresolved\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The chromatin-level mechanism was undefined; showing GRA28 boosts chromatin accessibility at the Ccr7 locus and enhances GRA15/GRA24/NF-κB-dependent transcription placed GRA28 within a cooperative effector module driving CCR7-mediated chemotaxis and dissemination.\",\n      \"evidence\": \"Combinatorial GRA knockouts, chromatin accessibility assays, in vivo competitive adoptive transfer in type II strain\",\n      \"pmids\": [\"39207098\", \"38370679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether GRA28 acts directly at the locus or via recruited factors unresolved\",\n        \"Biochemical partners at the chromatin level not identified\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"GRA28's role in immune-driven cell death was unknown; identifying it among nuclear MYR1-dependent effectors that block IFNγ-driven host cell death, acting by disrupting CBP/p300 acetyltransferase activity and IFNγ-driven transcription, assigned it a direct enzymatic-interference mechanism.\",\n      \"evidence\": \"Genome-wide loss-of-function screens in IFNγ-primed human cells, combinatorial GRA knockouts, transcription and CBP/p300 acetyltransferase activity assays\",\n      \"pmids\": [\"39292011\", \"38234811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct physical interaction between GRA28 and CBP/p300 not established\",\n        \"Redundancy with other nuclear effectors complicates isolating GRA28's specific contribution\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"How GRA28 fits into host NF-κB signaling was incompletely mapped; placing it as one of eight MYR1-dependent effectors that additively activate the non-canonical NF-κB (RelB/p52) pathway upstream of TRAF3 depletion and NIK stabilization defined its network-level position.\",\n      \"evidence\": \"Combinatorial knockout panel, RelB/p52 nuclear accumulation assays, epistasis with MYR1 deletion in type I and type II strains (preprint)\",\n      \"pmids\": [\"41959509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint, not yet peer-reviewed\",\n        \"GRA28's individual contribution within the additive network not isolated\",\n        \"Direct molecular target upstream of TRAF3 depletion unknown\"\n      ],\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct host molecular target(s) of GRA28 and whether it binds chromatin, CBP/p300, or NF-κB components directly remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No demonstrated direct physical interaction partner for GRA28\",\n        \"No structural model or defined functional domains\",\n        \"Mechanism by which GRA28 alters chromatin accessibility is unresolved\"\n      ],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140713\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0140416\", \"supporting_discovery_ids\": [1, 3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0042025\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0038061\", \"supporting_discovery_ids\": [5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]},\n      {\"term_id\": \"GO:0002526\", \"supporting_discovery_ids\": [1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MYR1 (translocon required for GRA28 host export)\",\n      \"TgPPM3C (phosphatase required for GRA28 export)\",\n      \"CBP/p300 (host histone acetyltransferase whose activity GRA28 disrupts)\",\n      \"GRA15, GRA24 (cooperating effectors in NF-κB-dependent transcription)\"\n    ],\n    \"other_free_text\": [\n      \"GRA28 drives CCL22 chemokine production and CCR7 upregulation, conferring dendritic cell-like hypermigration on parasitized macrophages and promoting dissemination.\",\n      \"GRA28 blocks IFNγ-driven host cell death as part of a nuclear MYR1-dependent effector set (with TgIST, GRA16, GRA24).\",\n      \"GRA28 is dispensable for in vitro growth, egress, and acute virulence in the RH strain.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_231960","name":"GRA28","product":"dense granule protein GRA28","uniprot_acc":"S8EZU9","chromosome":"TGME49_chrVIII","start_pos":1670549},{"strain":"GT1","gene_id":"TGGT1_231960","name":null,"product":"putative omega secalin","uniprot_acc":null,"chromosome":"TGGT1_chrVIII","start_pos":1668838},{"strain":"RH88","gene_id":"TGRH88_076030","name":null,"product":"putative omega secalin","uniprot_acc":null,"chromosome":"CM023089","start_pos":6779627},{"strain":"VEG","gene_id":"TGVEG_231960B","name":null,"product":"putative omega secalin","uniprot_acc":"V4ZUL9","chromosome":"TGVEG_chrVIII","start_pos":1677225}]}