{"gene":"TG_294330","gene_symbol":"AMA4","run_date":"2026-09-10T21:09:19","timeline":{"discoveries":[{"year":2014,"finding":"AMA1 gene disruption in T. gondii results in upregulation of AMA1 homologues (including AMA4) and RON2 homologues that cooperate to support residual invasion, demonstrating molecular plasticity at the moving junction and functional redundancy among AMA-RON pairs.","method":"Reverse genetics (gene disruption), invasion assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic disruption with invasion phenotype readout; AMA4 involvement inferred from upregulation data in a single lab study without direct AMA4 knockout","pmids":["24934579"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgAMA4 forms a high-affinity functional complex with its coevolved partner TgRON2L1. Crystal structures of TgAMA4 in apo and TgRON2L1-bound forms revealed that TgAMA4 lacks both the deep surface groove and the key surface loop that govern RON2 ligand-binding selectivity in canonical AMAs, defining an unexpected architecture and assembly mechanism. Alanine scanning mutagenesis confirmed the functional interface. TgAMA4 phylogenetically clusters with Plasmodium MAEBL proteins and is expressed in sporozoites.","method":"X-ray crystallography (apo and ligand-bound structures), alanine scanning mutagenesis, phylogenetic analysis, binding affinity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structures of both apo and complex forms combined with mutagenesis in a single rigorous study","pmids":["26712012"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"AMA4 is expressed in bradyzoites and forms part of the moving junction during bradyzoite host cell invasion together with its partner RON2L1, functionally replacing the tachyzoite AMA1-RON2 pair. Conditional loss-of-function of AMA4 demonstrated its key role in cyst burden during the onset of chronic infection while being dispensable for the acute phase. Immunization with the AMA4-RON2L1 complex provided selective protection against the chronic form of toxoplasmosis.","method":"Reverse genetics (gene knockout/conditional knockdown), invasion assays, mouse infection model (acute and chronic), immunization/vaccination experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with defined in vivo phenotypic readout (cyst burden), multiple orthogonal methods (invasion assays, mouse model, vaccination), single lab but highly rigorous","pmids":["36693095"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgAMA4 is a divergent apical membrane antigen expressed in sporozoites and bradyzoites that forms a high-affinity complex with its coevolved partner TgRON2L1 at the moving junction; structural analysis revealed a unique binding architecture lacking the canonical deep groove and surface loop of other AMAs, and genetic loss-of-function established that the AMA4-RON2L1 pair drives host cell invasion specifically during the bradyzoite stage, with a key role in cyst burden during chronic infection while being dispensable for acute infection."},"narrative":{"mechanistic_narrative":"TgAMA4 is a divergent apical membrane antigen of Toxoplasma gondii that drives host cell invasion at the moving junction during specific life-cycle stages by pairing with its coevolved rhoptry-neck partner TgRON2L1 [PMID:26712012, PMID:36693095]. It was first implicated as a functionally redundant member of the AMA family: disruption of AMA1 upregulates AMA4 and RON2 homologues that together sustain residual invasion, revealing molecular plasticity at the moving junction [PMID:24934579]. TgAMA4 forms a high-affinity complex with TgRON2L1, and crystal structures of apo and ligand-bound forms show it lacks both the deep surface groove and the surface loop that govern RON2 selectivity in canonical AMAs, defining an unexpected binding architecture; the interface was mapped by alanine scanning, and TgAMA4 clusters phylogenetically with Plasmodium MAEBL proteins [PMID:26712012]. Functionally, AMA4 and RON2L1 constitute the moving junction of the bradyzoite stage, replacing the tachyzoite AMA1-RON2 pair, and conditional loss of AMA4 reduces cyst burden at the onset of chronic infection while remaining dispensable for the acute phase; immunization with the AMA4-RON2L1 complex confers selective protection against chronic toxoplasmosis [PMID:36693095].","teleology":[{"year":2014,"claim":"It was unknown whether the AMA1-RON2 moving junction was uniquely essential; disruption of AMA1 revealed that AMA4 and other AMA-RON homologues can be upregulated to support residual invasion, establishing functional redundancy and plasticity at the moving junction.","evidence":"Reverse genetics (AMA1 gene disruption) with invasion-assay readout in T. gondii","pmids":["24934579"],"confidence":"Medium","gaps":["AMA4 involvement was inferred from upregulation without a direct AMA4 knockout","Did not establish the native stage or context in which AMA4 normally functions","No direct demonstration of an AMA4-RON pairing"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"The molecular basis of AMA4-RON2 pairing was unknown; crystal structures of TgAMA4 apo and TgRON2L1-bound forms plus mutagenesis defined a high-affinity complex with an unexpected architecture lacking the canonical AMA groove and surface loop, showing AMA4 uses a distinct assembly mechanism.","evidence":"X-ray crystallography of apo and ligand-bound complex, alanine-scanning mutagenesis, binding-affinity and phylogenetic analysis in T. gondii","pmids":["26712012"],"confidence":"High","gaps":["In vivo function and stage-specific role were not established at this point","Structural work was based on sporozoite expression; bradyzoite relevance not yet shown","Basis of coevolution with RON2L1 not functionally tested in infection"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"The physiological role of AMA4 was unresolved; conditional loss-of-function showed AMA4-RON2L1 forms the bradyzoite moving junction, replacing the tachyzoite AMA1-RON2 pair, and is required for cyst burden during chronic but not acute infection, with the complex serving as a stage-selective vaccine antigen.","evidence":"Conditional knockout/knockdown, invasion assays, acute/chronic mouse infection models, and immunization experiments in T. gondii","pmids":["36693095"],"confidence":"High","gaps":["Mechanism of stage-specific AMA4/RON2L1 expression switching not defined","Whether residual acute-phase invasion uses other AMA-RON pairs not resolved here","Structural basis of the bradyzoite junction in the intact parasite not shown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How AMA4-RON2L1 expression is restricted to the bradyzoite (and sporozoite) stages, and how the moving-junction hand-off between AMA-RON pairs is regulated across the life cycle, remains unresolved in the available corpus.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No transcriptional/regulatory mechanism for stage-specific AMA4 expression identified","No structural model of the AMA4 junction in situ during invasion","Full complement of AMA4-interacting rhoptry and micronemal proteins not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["TgAMA4-TgRON2L1 moving junction complex"],"partners":["TGRON2L1"],"other_free_text":["TgAMA4 is a divergent apical membrane antigen expressed in sporozoites and bradyzoites; phylogenetically clusters with Plasmodium MAEBL proteins [#1].","Crystal structures show TgAMA4 lacks the deep surface groove and surface loop governing RON2 selectivity in canonical AMAs, defining a distinct binding architecture [#1].","AMA4-RON2L1 functionally replaces the tachyzoite AMA1-RON2 pair at the bradyzoite moving junction and is required for cyst burden in chronic but not acute infection [#2].","Immunization with the AMA4-RON2L1 complex confers selective protection against chronic toxoplasmosis [#2]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"24934579","id":"PMC_24934579","title":"Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/24934579","citation_count":128,"is_preprint":false},{"pmid":"18182087","id":"PMC_18182087","title":"Infection with Toxoplasma gondii results in dysregulation of the host cell cycle.","date":"2008","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/18182087","citation_count":70,"is_preprint":false},{"pmid":"26712012","id":"PMC_26712012","title":"Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA-RON2 pair.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26712012","citation_count":29,"is_preprint":false},{"pmid":"36693095","id":"PMC_36693095","title":"Invasion of Toxoplasma gondii bradyzoites: Molecular dissection of the moving junction proteins and effective vaccination targets.","date":"2023","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/36693095","citation_count":24,"is_preprint":false},{"pmid":"23721065","id":"PMC_23721065","title":"Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells.","date":"2013","source":"BMC microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23721065","citation_count":18,"is_preprint":false},{"pmid":"30535987","id":"PMC_30535987","title":"Resveratrol-mediated reversal of changes in purinergic signaling and immune response induced by Toxoplasma gondii infection of neural progenitor cells.","date":"2018","source":"Purinergic signalling","url":"https://pubmed.ncbi.nlm.nih.gov/30535987","citation_count":15,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10362,"output_tokens":1191,"usd":0.024476,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5932,"output_tokens":2376,"usd":0.04453,"stage2_stop_reason":"end_turn"},"total_usd":0.069006,"stage1_batch_id":"msgbatch_015xmQKDRqnTh9anE4m7LiD6","stage2_batch_id":"msgbatch_01JLpkFTVhoQBNgqHCKFrVVU","note":"batch pricing = 50% of standard","corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"paper list: removed papers about other genes"},"stage1_raw":"Looking at the corpus, I need to identify papers specifically about AMA4 (TgAMA4) in Toxoplasma gondii, not other proteins like MIC1, MIC3, MIC6, etc.\n\nPapers directly about AMA4:\n- [3] PMID:24934579 — mentions AMA4 as upregulated when AMA1 is disrupted\n- [8] PMID:26712012 — directly characterizes TgAMA4 structure and its interaction with TgRON2L1\n- [10] PMID:36693095 — directly characterizes AMA4's role in bradyzoite invasion\n\nAll other papers are about MIC proteins (MIC1, MIC3, MIC6, etc.) and are not about AMA4. I will exclude them.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"AMA1 gene disruption in T. gondii results in upregulation of AMA1 homologues (including AMA4) and RON2 homologues that cooperate to support residual invasion, demonstrating molecular plasticity at the moving junction and functional redundancy among AMA-RON pairs.\",\n      \"method\": \"Reverse genetics (gene disruption), invasion assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic disruption with invasion phenotype readout; AMA4 involvement inferred from upregulation data in a single lab study without direct AMA4 knockout\",\n      \"pmids\": [\"24934579\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgAMA4 forms a high-affinity functional complex with its coevolved partner TgRON2L1. Crystal structures of TgAMA4 in apo and TgRON2L1-bound forms revealed that TgAMA4 lacks both the deep surface groove and the key surface loop that govern RON2 ligand-binding selectivity in canonical AMAs, defining an unexpected architecture and assembly mechanism. Alanine scanning mutagenesis confirmed the functional interface. TgAMA4 phylogenetically clusters with Plasmodium MAEBL proteins and is expressed in sporozoites.\",\n      \"method\": \"X-ray crystallography (apo and ligand-bound structures), alanine scanning mutagenesis, phylogenetic analysis, binding affinity assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structures of both apo and complex forms combined with mutagenesis in a single rigorous study\",\n      \"pmids\": [\"26712012\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AMA4 is expressed in bradyzoites and forms part of the moving junction during bradyzoite host cell invasion together with its partner RON2L1, functionally replacing the tachyzoite AMA1-RON2 pair. Conditional loss-of-function of AMA4 demonstrated its key role in cyst burden during the onset of chronic infection while being dispensable for the acute phase. Immunization with the AMA4-RON2L1 complex provided selective protection against the chronic form of toxoplasmosis.\",\n      \"method\": \"Reverse genetics (gene knockout/conditional knockdown), invasion assays, mouse infection model (acute and chronic), immunization/vaccination experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with defined in vivo phenotypic readout (cyst burden), multiple orthogonal methods (invasion assays, mouse model, vaccination), single lab but highly rigorous\",\n      \"pmids\": [\"36693095\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgAMA4 is a divergent apical membrane antigen expressed in sporozoites and bradyzoites that forms a high-affinity complex with its coevolved partner TgRON2L1 at the moving junction; structural analysis revealed a unique binding architecture lacking the canonical deep groove and surface loop of other AMAs, and genetic loss-of-function established that the AMA4-RON2L1 pair drives host cell invasion specifically during the bradyzoite stage, with a key role in cyst burden during chronic infection while being dispensable for acute infection.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgAMA4 is a divergent apical membrane antigen of Toxoplasma gondii that drives host cell invasion at the moving junction during specific life-cycle stages by pairing with its coevolved rhoptry-neck partner TgRON2L1 [#1, #2]. It was first implicated as a functionally redundant member of the AMA family: disruption of AMA1 upregulates AMA4 and RON2 homologues that together sustain residual invasion, revealing molecular plasticity at the moving junction [#0]. TgAMA4 forms a high-affinity complex with TgRON2L1, and crystal structures of apo and ligand-bound forms show it lacks both the deep surface groove and the surface loop that govern RON2 selectivity in canonical AMAs, defining an unexpected binding architecture; the interface was mapped by alanine scanning, and TgAMA4 clusters phylogenetically with Plasmodium MAEBL proteins [#1]. Functionally, AMA4 and RON2L1 constitute the moving junction of the bradyzoite stage, replacing the tachyzoite AMA1-RON2 pair, and conditional loss of AMA4 reduces cyst burden at the onset of chronic infection while remaining dispensable for the acute phase; immunization with the AMA4-RON2L1 complex confers selective protection against chronic toxoplasmosis [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"It was unknown whether the AMA1-RON2 moving junction was uniquely essential; disruption of AMA1 revealed that AMA4 and other AMA-RON homologues can be upregulated to support residual invasion, establishing functional redundancy and plasticity at the moving junction.\",\n      \"evidence\": \"Reverse genetics (AMA1 gene disruption) with invasion-assay readout in T. gondii\",\n      \"pmids\": [\"24934579\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"AMA4 involvement was inferred from upregulation without a direct AMA4 knockout\",\n        \"Did not establish the native stage or context in which AMA4 normally functions\",\n        \"No direct demonstration of an AMA4-RON pairing\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The molecular basis of AMA4-RON2 pairing was unknown; crystal structures of TgAMA4 apo and TgRON2L1-bound forms plus mutagenesis defined a high-affinity complex with an unexpected architecture lacking the canonical AMA groove and surface loop, showing AMA4 uses a distinct assembly mechanism.\",\n      \"evidence\": \"X-ray crystallography of apo and ligand-bound complex, alanine-scanning mutagenesis, binding-affinity and phylogenetic analysis in T. gondii\",\n      \"pmids\": [\"26712012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo function and stage-specific role were not established at this point\",\n        \"Structural work was based on sporozoite expression; bradyzoite relevance not yet shown\",\n        \"Basis of coevolution with RON2L1 not functionally tested in infection\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The physiological role of AMA4 was unresolved; conditional loss-of-function showed AMA4-RON2L1 forms the bradyzoite moving junction, replacing the tachyzoite AMA1-RON2 pair, and is required for cyst burden during chronic but not acute infection, with the complex serving as a stage-selective vaccine antigen.\",\n      \"evidence\": \"Conditional knockout/knockdown, invasion assays, acute/chronic mouse infection models, and immunization experiments in T. gondii\",\n      \"pmids\": [\"36693095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism of stage-specific AMA4/RON2L1 expression switching not defined\",\n        \"Whether residual acute-phase invasion uses other AMA-RON pairs not resolved here\",\n        \"Structural basis of the bradyzoite junction in the intact parasite not shown\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AMA4-RON2L1 expression is restricted to the bradyzoite (and sporozoite) stages, and how the moving-junction hand-off between AMA-RON pairs is regulated across the life cycle, remains unresolved in the available corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No transcriptional/regulatory mechanism for stage-specific AMA4 expression identified\",\n        \"No structural model of the AMA4 junction in situ during invasion\",\n        \"Full complement of AMA4-interacting rhoptry and micronemal proteins not mapped\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005515\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"TgAMA4-TgRON2L1 moving junction complex\"\n    ],\n    \"partners\": [\n      \"TgRON2L1\"\n    ],\n    \"other_free_text\": [\n      \"TgAMA4 is a divergent apical membrane antigen expressed in sporozoites and bradyzoites; phylogenetically clusters with Plasmodium MAEBL proteins [#1].\",\n      \"Crystal structures show TgAMA4 lacks the deep surface groove and surface loop governing RON2 selectivity in canonical AMAs, defining a distinct binding architecture [#1].\",\n      \"AMA4-RON2L1 functionally replaces the tachyzoite AMA1-RON2 pair at the bradyzoite moving junction and is required for cyst burden in chronic but not acute infection [#2].\",\n      \"Immunization with the AMA4-RON2L1 complex confers selective protection against chronic toxoplasmosis [#2].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_294330","name":"AMA4","product":"apical membrane antigen AMA4","uniprot_acc":"A0A125YRE5","chromosome":"TGME49_chrIa","start_pos":802537},{"strain":"GT1","gene_id":"TGGT1_294330","name":null,"product":"EGF family domain-containing protein","uniprot_acc":null,"chromosome":"TGGT1_chrIa","start_pos":788197},{"strain":"RH88","gene_id":"TGRH88_019420","name":null,"product":"EGF family domain-containing protein","uniprot_acc":null,"chromosome":"CM023081","start_pos":808374},{"strain":"VEG","gene_id":"TGVEG_294330","name":null,"product":"EGF family domain-containing protein","uniprot_acc":"A0A125YRE7","chromosome":"TGVEG_chrIa","start_pos":811815}]}