{"gene":"TG_239740","gene_symbol":"GRA14","run_date":"2026-09-08T12:58:24","timeline":{"discoveries":[{"year":2008,"finding":"GRA14 is a novel dense granule protein secreted into the parasitophorous vacuole (PV) that localizes to the intravacuolar network (IVN) and the PV membrane (PVM). It has an unexpected transmembrane topology with its C-terminus facing the host cytoplasm and its N-terminus facing the vacuolar lumen.","method":"Subcellular fractionation, immunofluorescence, and protease protection assays; GRA14 knockout strain generated and used in coinfection experiments","journal":"Infection and immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiments with protease protection topology assay, knockout strain validation, and coinfection transfer assay, all in a single focused study","pmids":["18765740"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"GRA14 can be transferred between vacuoles (intervacuolar transport): wild-type parasites can donate GRA14 to GRA14-knockout vacuoles in coinfection experiments, demonstrating its mobility between PVs.","method":"Coinfection experiment using GRA14 knockout strain and wild-type parasites, immunofluorescence to detect GRA14 transfer","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, direct coinfection assay with knockout controls and fluorescence readout","pmids":["18765740"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"Deletion of PVM-associated GRA14 in the type II Prugniaud strain causes severe defects in chronic-stage cyst development in vivo without affecting in vitro growth rate or in vitro cyst differentiation, establishing a role for GRA14 in establishment of chronic infection.","method":"Genetic deletion (knockout) of GRA14 in Prugniaud strain, in vivo mouse infection model, in vitro cyst formation assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with specific in vivo chronic-infection phenotype, confirmed negative for in vitro growth defect, replicated across multiple GRA knockouts in same study","pmids":["31266861"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}]},{"year":2020,"finding":"GRA14 is partially involved in activation of the NF-κB pathway during T. gondii infection. Deletion of GRA14 in the type II Pru strain reduces nuclear translocation of RelA and decreases IL-6 production in macrophages, as well as downregulating NF-κB-mediated gene expression.","method":"Luciferase reporter NF-κB assay, GRA14 knockout in Pru strain, nuclear translocation assay for RelA, ELISA for IL-6, RNA-seq","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, multiple orthogonal methods (reporter assay, knockout, nuclear translocation, transcriptomics), but partial/incomplete effect","pmids":["32849602"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}]},{"year":2021,"finding":"TgGRA14 recruits host ESCRT machinery to the PV membrane via late-domain-like motifs homologous to those of HIV-1 Gag. The motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent virus-like particle (VLP) budding. TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins. GRA14-deficient parasites show a marked reduction in uptake of host cytosolic proteins.","method":"HIV-1 VLP release assay (functional substitution), proximity ligation/co-IP with host ESCRT components, GRA14 knockout parasites with host cytosolic protein ingestion assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods: functional ESCRT substitution assay, interaction studies, and loss-of-function with specific ingestion phenotype, all in a single focused study","pmids":["34898650"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"TgGRA14 (along with TgGRA64) interacts with ESCRT components and is required for ESCRT-driven PVM invaginations and scavenging of host organelles into the PV. Parasites lacking GRA14 showed reduced CHMP4B-dominant-negative-induced PVM invaginations and fewer intra-PV host organelles, with greater defects in a GRA14/GRA64 double knockout.","method":"Dominant-negative ESCRT constructs (DN-CHMP4B, DN-Vps4A), GRA14 and GRA64 single and double knockouts, fluorescence microscopy of PVM invaginations and intra-PV host organelle accumulation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — single and double knockout analysis with dominant-negative ESCRT tools and multiple microscopy readouts, independently replicating and extending the ESCRT-GRA14 interaction","pmids":["36718630"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"The ingestion pathway initiated by GRA14 contributes to nutrient scavenging: Δgra14 parasites, like Δcpl and Δcrt mutants, show reduced glycolytic intermediates and amino acids and an exacerbated growth defect under amino acid-limiting conditions, consistent with GRA14-driven ESCRT recruitment being the initiating step of a pathway that delivers host cytosolic nutrients to the parasite.","method":"Genome-wide CRISPR fitness screen in WT and Δgra14/Δcpl/Δcrt mutants, bulk metabolomics, growth assay in amino acid-depleted media","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR genome-wide screen plus metabolomics in single study, but indirect evidence for GRA14's nutrient role (measured downstream mutants); single lab","pmids":["40172222"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"CRISPR-Cas9-mediated deletion of GRA14 in the virulent type I RH strain had no significant effect on in vitro growth, egress, or acute virulence in mice, indicating GRA14 is dispensable for these processes in this strain.","method":"CRISPR-Cas9 gene disruption, plaque assay, egress assay, mouse virulence model","journal":"Frontiers in cellular and infection microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR knockout with multiple in vitro and in vivo phenotypic readouts, but negative finding (no effect in RH strain); single lab","pmids":["30211128"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"GRA14 is a transmembrane dense granule protein that localizes to the PVM and IVN with its C-terminus exposed to the host cytoplasm; it initiates an ingestion pathway by recruiting host ESCRT machinery (via late-domain-like motifs) to the PVM, driving membrane invagination and vesicular uptake of host cytosolic proteins and organelles into the PV for nutrient acquisition, while also partially modulating host NF-κB signaling and being required for chronic cyst development in vivo."},"narrative":{"mechanistic_narrative":"GRA14 is a Toxoplasma gondii dense granule protein secreted into the parasitophorous vacuole, where it adopts a transmembrane topology at the PV membrane (PVM) and intravacuolar network (IVN) with its C-terminus exposed to the host cytoplasm and its N-terminus in the vacuolar lumen [PMID:18765740]. Through late-domain-like motifs homologous to those of HIV-1 Gag, GRA14 recruits host ESCRT machinery to the PVM—its motif-containing region can functionally substitute for the HIV-1 Gag late domain in ESCRT-dependent virus-like particle budding—and it interacts with host ESCRT components at the vacuole [PMID:34898650]. This ESCRT recruitment drives PVM invaginations and the vesicular uptake of host cytosolic proteins and organelles into the PV, an activity that GRA14 shares with GRA64 and that is more severely impaired in a double knockout [PMID:34898650, PMID:36718630]. The resulting ingestion pathway contributes to nutrient scavenging, as Δgra14 parasites show reduced glycolytic intermediates and amino acids and a growth defect under amino acid limitation [PMID:40172222]. GRA14 is also partially required for activation of host NF-κB signaling, promoting RelA nuclear translocation and IL-6 production in infected macrophages [PMID:32849602], and is required in vivo for chronic-stage cyst development [PMID:31266861]. GRA14 can be transferred between vacuoles during coinfection, indicating mobility between PVs [PMID:18765740].","teleology":[{"year":2008,"claim":"Before its topology was defined, it was unknown how a dense granule protein could act at the host-parasite interface; this work established GRA14 as a PVM/IVN transmembrane protein with its C-terminus facing the host cytoplasm, positioning it to engage host machinery.","evidence":"Subcellular fractionation, immunofluorescence, and protease protection topology assays with a GRA14 knockout strain","pmids":["18765740"],"confidence":"High","gaps":["Host-facing binding partners not identified at this stage","Functional consequence of the topology unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"It was unclear whether dense granule proteins are static within their vacuole; coinfection transfer of GRA14 from wild-type to knockout vacuoles demonstrated intervacuolar mobility.","evidence":"Coinfection of GRA14 knockout and wild-type parasites with immunofluorescence detection of transferred GRA14","pmids":["18765740"],"confidence":"Medium","gaps":["Mechanism and route of transfer between PVs unknown","Functional relevance of transfer not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"To test whether GRA14 is required for acute infection, its deletion in the virulent type I RH strain was assessed; loss had no effect on in vitro growth, egress, or acute virulence, showing GRA14 is dispensable for these processes in this strain.","evidence":"CRISPR-Cas9 disruption in RH with plaque, egress, and mouse virulence assays","pmids":["30211128"],"confidence":"Medium","gaps":["Negative finding restricted to RH acute infection","Chronic-stage role not addressed in this strain"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"claim":"The in vivo role of GRA14 was unresolved; deletion in the type II Prugniaud strain revealed a specific defect in chronic-stage cyst development without affecting in vitro growth or differentiation, establishing GRA14 in chronic infection.","evidence":"GRA14 knockout in Prugniaud with in vivo mouse infection and in vitro cyst formation assays","pmids":["31266861"],"confidence":"High","gaps":["Molecular basis linking GRA14 to cyst persistence unresolved","Relationship to its PVM/ingestion functions not established"],"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}]},{"year":2020,"claim":"Whether GRA14 modulates host innate signaling was unknown; its deletion in Pru reduced RelA nuclear translocation, IL-6 production, and NF-κB-driven transcription, implicating GRA14 partially in host NF-κB activation.","evidence":"NF-κB luciferase reporter, GRA14 knockout in Pru, RelA nuclear translocation assay, IL-6 ELISA, and RNA-seq in macrophages","pmids":["32849602"],"confidence":"Medium","gaps":["Effect is partial and mechanism of NF-κB activation unresolved","Direct versus indirect involvement not distinguished"],"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}]},{"year":2021,"claim":"How the parasite co-opts host membrane-remodeling machinery was unknown; GRA14 was shown to recruit host ESCRT via HIV-1 Gag-like late-domain motifs and to be required for host cytosolic protein ingestion, defining a molecular mechanism for host-material uptake.","evidence":"HIV-1 VLP release functional substitution assay, proximity/co-IP with host ESCRT components, and GRA14 knockout ingestion assay","pmids":["34898650"],"confidence":"High","gaps":["Precise ESCRT subunit engaged by the late-domain motif not fully mapped","Stoichiometry with other dense granule proteins unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"It was unclear whether ESCRT recruitment drives PVM membrane deformation and organelle uptake; GRA14 (with GRA64) was shown required for ESCRT-driven PVM invaginations and scavenging of host organelles, with a stronger double-knockout defect.","evidence":"Dominant-negative CHMP4B and Vps4A constructs, GRA14/GRA64 single and double knockouts, and microscopy of PVM invaginations and intra-PV organelles","pmids":["36718630"],"confidence":"High","gaps":["Functional division of labor between GRA14 and GRA64 not resolved","Selectivity of organelle capture unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Whether the GRA14-initiated ingestion pathway feeds parasite metabolism was open; Δgra14 parasites showed reduced glycolytic intermediates and amino acids and a growth defect under amino acid limitation, linking GRA14-driven ESCRT recruitment to host nutrient scavenging.","evidence":"Genome-wide CRISPR fitness screen, bulk metabolomics, and growth assays in amino acid-depleted media in WT and Δgra14/Δcpl/Δcrt mutants","pmids":["40172222"],"confidence":"Medium","gaps":["Evidence for GRA14's nutrient role is partly inferred from downstream mutants","Specific nutrients delivered via the pathway not directly demonstrated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How GRA14's transmembrane topology, ESCRT-recruiting ingestion function, NF-κB modulation, and requirement for chronic cyst development are mechanistically integrated remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No mechanism links GRA14-driven ingestion to chronic cyst persistence","The host receptor/ESCRT subunit specificity of the late-domain motif is not fully defined","Direct role in NF-κB signaling versus indirect consequence is unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Host ESCRT machinery (via GRA14 late-domain-like motifs)"],"partners":["HOST ESCRT COMPONENTS","CHMP4B (ESCRT-III)","VPS4A","TGGRA64","HIV-1 GAG LATE DOMAIN (FUNCTIONAL SUBSTITUTION)"],"other_free_text":["Transmembrane dense granule protein with C-terminus in host cytoplasm and N-terminus in vacuolar lumen [#0]","Mobile between parasitophorous vacuoles via intervacuolar transfer [#1]","Required for chronic-stage cyst development in vivo in type II Pru, but dispensable for acute virulence in type I RH","Initiates ESCRT-dependent ingestion of host cytosolic proteins and organelles contributing to nutrient scavenging [#4, #5, #6]"]},"strain_conflicts":[{"aspect":"Requirement of GRA14 for in vivo infection","claims":[{"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}],"claim":"GRA14 deletion has no effect on in vitro growth, egress, or acute virulence, indicating it is dispensable for acute infection in the type I RH strain","supporting_discovery_ids":[7]},{"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}],"claim":"GRA14 deletion causes severe defects in chronic-stage cyst development in vivo without affecting in vitro growth, showing a requirement for establishing chronic infection in the type II Pru strain","supporting_discovery_ids":[2]}]}]},"prefetch_data":{},"corpus_meta":[{"pmid":"18765740","id":"PMC_18765740","title":"Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii.","date":"2008","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/18765740","citation_count":91,"is_preprint":false},{"pmid":"31266861","id":"PMC_31266861","title":"Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon.","date":"2019","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/31266861","citation_count":75,"is_preprint":false},{"pmid":"23408523","id":"PMC_23408523","title":"Development of an immunochromatographic assay based on dense granule protein 7 for serological detection of Toxoplasma gondii infection.","date":"2013","source":"Clinical and vaccine immunology : CVI","url":"https://pubmed.ncbi.nlm.nih.gov/23408523","citation_count":60,"is_preprint":false},{"pmid":"34898650","id":"PMC_34898650","title":"Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins.","date":"2021","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/34898650","citation_count":44,"is_preprint":false},{"pmid":"28286231","id":"PMC_28286231","title":"Enhancing immune responses to a DNA vaccine encoding Toxoplasma gondii GRA14 by calcium phosphate nanoparticles as an adjuvant.","date":"2017","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/28286231","citation_count":39,"is_preprint":false},{"pmid":"32849602","id":"PMC_32849602","title":"Toxoplasma gondii Dense Granule Proteins 7, 14, and 15 Are Involved in Modification and Control of the Immune Response Mediated via NF-κB Pathway.","date":"2020","source":"Frontiers in 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Philippines.","date":"2022","source":"Pathogens (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/35215219","citation_count":8,"is_preprint":false},{"pmid":"34801755","id":"PMC_34801755","title":"Evaluation of Th17 immune responses of recombinant DNA vaccine encoding GRA14 and ROP13 genes against Toxoplasma gondii in BALB/c mice.","date":"2021","source":"Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/34801755","citation_count":7,"is_preprint":false},{"pmid":"40172222","id":"PMC_40172222","title":"Metabolic adaptability and nutrient scavenging in Toxoplasma gondii: insights from ingestion pathway-deficient mutants.","date":"2025","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/40172222","citation_count":6,"is_preprint":false},{"pmid":"29963084","id":"PMC_29963084","title":"Identification of Antigenic and Immunogenic Proteins of Toxoplasma gondii in Human and Sheep by Immunoproteomics.","date":"2018","source":"Iranian journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/29963084","citation_count":6,"is_preprint":false},{"pmid":"39651188","id":"PMC_39651188","title":"Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39651188","citation_count":1,"is_preprint":false},{"pmid":"34509125","id":"PMC_34509125","title":"A PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene.","date":"2021","source":"Veterinary parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/34509125","citation_count":1,"is_preprint":false},{"pmid":"41599200","id":"PMC_41599200","title":"Protection Against Toxoplasma gondii Lethal ME49 Challenge Induced by Influenza Virus-like Particles Containing Dense Granule Protein 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It has an unexpected transmembrane topology with its C-terminus facing the host cytoplasm and its N-terminus facing the vacuolar lumen.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence, and protease protection assays; GRA14 knockout strain generated and used in coinfection experiments\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiments with protease protection topology assay, knockout strain validation, and coinfection transfer assay, all in a single focused study\",\n      \"pmids\": [\"18765740\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GRA14 can be transferred between vacuoles (intervacuolar transport): wild-type parasites can donate GRA14 to GRA14-knockout vacuoles in coinfection experiments, demonstrating its mobility between PVs.\",\n      \"method\": \"Coinfection experiment using GRA14 knockout strain and wild-type parasites, immunofluorescence to detect GRA14 transfer\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, direct coinfection assay with knockout controls and fluorescence readout\",\n      \"pmids\": [\"18765740\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deletion of PVM-associated GRA14 in the type II Prugniaud strain causes severe defects in chronic-stage cyst development in vivo without affecting in vitro growth rate or in vitro cyst differentiation, establishing a role for GRA14 in establishment of chronic infection.\",\n      \"method\": \"Genetic deletion (knockout) of GRA14 in Prugniaud strain, in vivo mouse infection model, in vitro cyst formation assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with specific in vivo chronic-infection phenotype, confirmed negative for in vitro growth defect, replicated across multiple GRA knockouts in same study\",\n      \"pmids\": [\"31266861\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA14 is partially involved in activation of the NF-κB pathway during T. gondii infection. Deletion of GRA14 in the type II Pru strain reduces nuclear translocation of RelA and decreases IL-6 production in macrophages, as well as downregulating NF-κB-mediated gene expression.\",\n      \"method\": \"Luciferase reporter NF-κB assay, GRA14 knockout in Pru strain, nuclear translocation assay for RelA, ELISA for IL-6, RNA-seq\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, multiple orthogonal methods (reporter assay, knockout, nuclear translocation, transcriptomics), but partial/incomplete effect\",\n      \"pmids\": [\"32849602\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TgGRA14 recruits host ESCRT machinery to the PV membrane via late-domain-like motifs homologous to those of HIV-1 Gag. The motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent virus-like particle (VLP) budding. TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins. GRA14-deficient parasites show a marked reduction in uptake of host cytosolic proteins.\",\n      \"method\": \"HIV-1 VLP release assay (functional substitution), proximity ligation/co-IP with host ESCRT components, GRA14 knockout parasites with host cytosolic protein ingestion assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods: functional ESCRT substitution assay, interaction studies, and loss-of-function with specific ingestion phenotype, all in a single focused study\",\n      \"pmids\": [\"34898650\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TgGRA14 (along with TgGRA64) interacts with ESCRT components and is required for ESCRT-driven PVM invaginations and scavenging of host organelles into the PV. Parasites lacking GRA14 showed reduced CHMP4B-dominant-negative-induced PVM invaginations and fewer intra-PV host organelles, with greater defects in a GRA14/GRA64 double knockout.\",\n      \"method\": \"Dominant-negative ESCRT constructs (DN-CHMP4B, DN-Vps4A), GRA14 and GRA64 single and double knockouts, fluorescence microscopy of PVM invaginations and intra-PV host organelle accumulation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — single and double knockout analysis with dominant-negative ESCRT tools and multiple microscopy readouts, independently replicating and extending the ESCRT-GRA14 interaction\",\n      \"pmids\": [\"36718630\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The ingestion pathway initiated by GRA14 contributes to nutrient scavenging: Δgra14 parasites, like Δcpl and Δcrt mutants, show reduced glycolytic intermediates and amino acids and an exacerbated growth defect under amino acid-limiting conditions, consistent with GRA14-driven ESCRT recruitment being the initiating step of a pathway that delivers host cytosolic nutrients to the parasite.\",\n      \"method\": \"Genome-wide CRISPR fitness screen in WT and Δgra14/Δcpl/Δcrt mutants, bulk metabolomics, growth assay in amino acid-depleted media\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR genome-wide screen plus metabolomics in single study, but indirect evidence for GRA14's nutrient role (measured downstream mutants); single lab\",\n      \"pmids\": [\"40172222\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRISPR-Cas9-mediated deletion of GRA14 in the virulent type I RH strain had no significant effect on in vitro growth, egress, or acute virulence in mice, indicating GRA14 is dispensable for these processes in this strain.\",\n      \"method\": \"CRISPR-Cas9 gene disruption, 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 knockout with multiple in vitro and in vivo phenotypic readouts, but negative finding (no effect in RH strain); single lab\",\n      \"pmids\": [\"30211128\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"GRA14 is a transmembrane dense granule protein that localizes to the PVM and IVN with its C-terminus exposed to the host cytoplasm; it initiates an ingestion pathway by recruiting host ESCRT machinery (via late-domain-like motifs) to the PVM, driving membrane invagination and vesicular uptake of host cytosolic proteins and organelles into the PV for nutrient acquisition, while also partially modulating host NF-κB signaling and being required for chronic cyst development in vivo.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GRA14 is a Toxoplasma gondii dense granule protein secreted into the parasitophorous vacuole, where it adopts a transmembrane topology at the PV membrane (PVM) and intravacuolar network (IVN) with its C-terminus exposed to the host cytoplasm and its N-terminus in the vacuolar lumen [#0]. Through late-domain-like motifs homologous to those of HIV-1 Gag, GRA14 recruits host ESCRT machinery to the PVM—its motif-containing region can functionally substitute for the HIV-1 Gag late domain in ESCRT-dependent virus-like particle budding—and it interacts with host ESCRT components at the vacuole [#4]. This ESCRT recruitment drives PVM invaginations and the vesicular uptake of host cytosolic proteins and organelles into the PV, an activity that GRA14 shares with GRA64 and that is more severely impaired in a double knockout [#4, #5]. The resulting ingestion pathway contributes to nutrient scavenging, as Δgra14 parasites show reduced glycolytic intermediates and amino acids and a growth defect under amino acid limitation [#6]. GRA14 is also partially required for activation of host NF-κB signaling, promoting RelA nuclear translocation and IL-6 production in infected macrophages [#3], and is required in vivo for chronic-stage cyst development [#2]. GRA14 can be transferred between vacuoles during coinfection, indicating mobility between PVs [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Before its topology was defined, it was unknown how a dense granule protein could act at the host-parasite interface; this work established GRA14 as a PVM/IVN transmembrane protein with its C-terminus facing the host cytoplasm, positioning it to engage host machinery.\",\n      \"evidence\": \"Subcellular fractionation, immunofluorescence, and protease protection topology assays with a GRA14 knockout strain\",\n      \"pmids\": [\"18765740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host-facing binding partners not identified at this stage\", \"Functional consequence of the topology unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"It was unclear whether dense granule proteins are static within their vacuole; coinfection transfer of GRA14 from wild-type to knockout vacuoles demonstrated intervacuolar mobility.\",\n      \"evidence\": \"Coinfection of GRA14 knockout and wild-type parasites with immunofluorescence detection of transferred GRA14\",\n      \"pmids\": [\"18765740\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism and route of transfer between PVs unknown\", \"Functional relevance of transfer not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"To test whether GRA14 is required for acute infection, its deletion in the virulent type I RH strain was assessed; loss had no effect on in vitro growth, egress, or acute virulence, showing GRA14 is dispensable for these processes in this strain.\",\n      \"evidence\": \"CRISPR-Cas9 disruption in RH with plaque, egress, and mouse virulence assays\",\n      \"pmids\": [\"30211128\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative finding restricted to RH acute infection\", \"Chronic-stage role not addressed in this strain\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The in vivo role of GRA14 was unresolved; deletion in the type II Prugniaud strain revealed a specific defect in chronic-stage cyst development without affecting in vitro growth or differentiation, establishing GRA14 in chronic infection.\",\n      \"evidence\": \"GRA14 knockout in Prugniaud with in vivo mouse infection and in vitro cyst formation assays\",\n      \"pmids\": [\"31266861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis linking GRA14 to cyst persistence unresolved\", \"Relationship to its PVM/ingestion functions not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether GRA14 modulates host innate signaling was unknown; its deletion in Pru reduced RelA nuclear translocation, IL-6 production, and NF-κB-driven transcription, implicating GRA14 partially in host NF-κB activation.\",\n      \"evidence\": \"NF-κB luciferase reporter, GRA14 knockout in Pru, RelA nuclear translocation assay, IL-6 ELISA, and RNA-seq in macrophages\",\n      \"pmids\": [\"32849602\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effect is partial and mechanism of NF-κB activation unresolved\", \"Direct versus indirect involvement not distinguished\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"How the parasite co-opts host membrane-remodeling machinery was unknown; GRA14 was shown to recruit host ESCRT via HIV-1 Gag-like late-domain motifs and to be required for host cytosolic protein ingestion, defining a molecular mechanism for host-material uptake.\",\n      \"evidence\": \"HIV-1 VLP release functional substitution assay, proximity/co-IP with host ESCRT components, and GRA14 knockout ingestion assay\",\n      \"pmids\": [\"34898650\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise ESCRT subunit engaged by the late-domain motif not fully mapped\", \"Stoichiometry with other dense granule proteins unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"It was unclear whether ESCRT recruitment drives PVM membrane deformation and organelle uptake; GRA14 (with GRA64) was shown required for ESCRT-driven PVM invaginations and scavenging of host organelles, with a stronger double-knockout defect.\",\n      \"evidence\": \"Dominant-negative CHMP4B and Vps4A constructs, GRA14/GRA64 single and double knockouts, and microscopy of PVM invaginations and intra-PV organelles\",\n      \"pmids\": [\"36718630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional division of labor between GRA14 and GRA64 not resolved\", \"Selectivity of organelle capture unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Whether the GRA14-initiated ingestion pathway feeds parasite metabolism was open; Δgra14 parasites showed reduced glycolytic intermediates and amino acids and a growth defect under amino acid limitation, linking GRA14-driven ESCRT recruitment to host nutrient scavenging.\",\n      \"evidence\": \"Genome-wide CRISPR fitness screen, bulk metabolomics, and growth assays in amino acid-depleted media in WT and Δgra14/Δcpl/Δcrt mutants\",\n      \"pmids\": [\"40172222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Evidence for GRA14's nutrient role is partly inferred from downstream mutants\", \"Specific nutrients delivered via the pathway not directly demonstrated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRA14's transmembrane topology, ESCRT-recruiting ingestion function, NF-κB modulation, and requirement for chronic cyst development are mechanistically integrated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mechanism links GRA14-driven ingestion to chronic cyst persistence\", \"The host receptor/ESCRT subunit specificity of the late-domain motif is not fully defined\", \"Direct role in NF-κB signaling versus indirect consequence is unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0035091\", \"supporting_discovery_ids\": [4, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006914\", \"supporting_discovery_ids\": [4, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0038061\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]}\n    ],\n    \"complexes\": [\"Host ESCRT machinery (via GRA14 late-domain-like motifs)\"],\n    \"partners\": [\"Host ESCRT components\", \"CHMP4B (ESCRT-III)\", \"Vps4A\", \"TgGRA64\", \"HIV-1 Gag late domain (functional substitution)\"],\n    \"other_free_text\": [\n      \"Transmembrane dense granule protein with C-terminus in host cytoplasm and N-terminus in vacuolar lumen [#0]\",\n      \"Mobile between parasitophorous vacuoles via intervacuolar transfer [#1]\",\n      \"Required for chronic-stage cyst development in vivo in type II Pru, but dispensable for acute virulence in type I RH\",\n      \"Initiates ESCRT-dependent ingestion of host cytosolic proteins and organelles contributing to nutrient scavenging [#4, #5, #6]\"\n    ]\n  },\n  \"strain_conflicts\": [\n    {\n      \"aspect\": \"Requirement of GRA14 for in vivo infection\",\n      \"claims\": [\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}], \"claim\": \"GRA14 deletion has no effect on in vitro growth, egress, or acute virulence, indicating it is dispensable for acute infection in the type I RH strain\", \"supporting_discovery_ids\": [7]},\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}], \"claim\": \"GRA14 deletion causes severe defects in chronic-stage cyst development in vivo without affecting in vitro growth, showing a requirement for establishing chronic infection in the type II Pru strain\", \"supporting_discovery_ids\": [2]}\n      ]\n    }\n  ]\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_239740","name":"GRA14","product":"dense granule protein GRA14","uniprot_acc":"A0A125YK96","chromosome":"TGME49_chrVI","start_pos":882305},{"strain":"GT1","gene_id":"TGGT1_239740","name":null,"product":"dense granule protein GRA14","uniprot_acc":null,"chromosome":"TGGT1_chrVI","start_pos":875718},{"strain":"RH88","gene_id":"TGRH88_031240","name":null,"product":"dense granule protein GRA14","uniprot_acc":null,"chromosome":"CM023087","start_pos":2819510},{"strain":"VEG","gene_id":"TGVEG_239740","name":null,"product":"dense granule protein GRA14","uniprot_acc":"B6KF82","chromosome":"TGVEG_chrVI","start_pos":895984}]}