{"gene":"TG_310670","gene_symbol":"GP","run_date":"2026-09-10T21:09:19","timeline":{"discoveries":[{"year":2017,"finding":"TgGP (glycogen phosphorylase) is required for amylopectin catabolism in Toxoplasma gondii bradyzoites; a stop codon mutant (S25stop) and a phosphorylation-null mutant (S25A) both display amylopectin accumulation, while a phosphorylation-mimetic mutant (S25E) shows minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions, establishing that phosphorylation of Ser25 activates TgGP to drive amylopectin digestion.","method":"Site-directed mutagenesis (S25stop, S25A, S25E) combined with CRISPR/genome editing, amylopectin staining, and complementation rescue experiments in T. gondii","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple separation-of-function mutants with complementation rescue, replicated across growth conditions in a single rigorous study","pmids":["28851850"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Pru"}]},{"year":2017,"finding":"Both loss of TgGP activity (S25A inactive mutant) and constitutive TgGP activity (S25E hyperactive mutant) significantly reduced brain cyst production in mice compared to wild-type, and complementation restoring normal amylopectin regulation restored cyst production to control levels, establishing that tight TgGP-mediated regulation of amylopectin is required for efficient cyst formation.","method":"Mouse infection model with cyst quantification, complementation experiment","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function and gain-of-function with complementation rescue, defined in vivo phenotypic readout in a single rigorous study","pmids":["28851850"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Pru"}]}],"current_model":"TgGP (putative glycogen phosphorylase) is the enzyme responsible for amylopectin catabolism in Toxoplasma gondii bradyzoites, regulated by phosphorylation of Ser25 (activated by phosphorylation, inactivated when dephosphorylated), acts downstream of CDPK2 in the amylopectin regulatory pathway, and requires tight activity control — neither constitutive inactivation nor constitutive activation — for efficient production of brain tissue cysts during chronic infection."},"narrative":{"mechanistic_narrative":"TgGP (putative glycogen phosphorylase) is the enzyme responsible for amylopectin catabolism in Toxoplasma gondii bradyzoites, and its activity is governed by phosphorylation of Ser25: a phosphorylation-null mutant (S25A) and a truncation mutant (S25stop) accumulate amylopectin, whereas a phosphomimetic mutant (S25E) shows minimal accumulation, establishing that Ser25 phosphorylation activates TgGP to drive amylopectin digestion [PMID:28851850]. Tight control of this activity is required for chronic infection, as both loss of activity (S25A) and constitutive activity (S25E) reduce brain cyst production in mice, while complementation restoring normal amylopectin regulation restores cyst formation [PMID:28851850]. Beyond this phosphoregulated role in amylopectin turnover, no further mechanistic detail about TgGP has been characterized in the available corpus.","teleology":[{"year":2017,"claim":"It was unknown which enzyme degrades the bradyzoite storage polysaccharide amylopectin and how it is controlled; separation-of-function mutants established that TgGP drives amylopectin catabolism and is switched on by Ser25 phosphorylation.","evidence":"Site-directed mutagenesis (S25stop, S25A, S25E) with CRISPR editing, amylopectin staining, and complementation rescue in T. gondii Pru","pmids":["28851850"],"confidence":"High","gaps":["The kinase and phosphatase acting on Ser25 are not identified in this finding","No direct biochemical demonstration of phosphorylase catalytic activity on amylopectin substrate"],"attribution":[{"species":"Toxoplasma gondii","strain":"Pru"}]},{"year":2017,"claim":"It was unknown whether the balance of amylopectin turnover matters for chronic infection; gain- and loss-of-function TgGP mutants showed that both constitutive inactivation and constitutive activation impair brain cyst formation, establishing that tight regulation of TgGP is required in vivo.","evidence":"Mouse infection model with cyst quantification and complementation rescue in T. gondii Pru","pmids":["28851850"],"confidence":"High","gaps":["Does not resolve how amylopectin flux mechanistically supports cyst maturation","Upstream regulatory signals controlling TgGP activity in vivo are not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"Pru"}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":[],"other_free_text":["Activity switched on by phosphorylation of Ser25 (S25E phosphomimetic active, S25A phosphonull inactive) [#0]","Tight regulation of TgGP-mediated amylopectin turnover required for efficient brain cyst formation in mice [#1]","Identified as a lysine-crotonylated protein in a global crotonylome catalog (Low confidence, unvalidated) [#2]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"28851850","id":"PMC_28851850","title":"Toxoplasma gondii Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts.","date":"2017","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/28851850","citation_count":50,"is_preprint":false},{"pmid":"32245165","id":"PMC_32245165","title":"The Bradyzoite: A Key Developmental Stage for the Persistence and Pathogenesis of Toxoplasmosis.","date":"2020","source":"Pathogens (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/32245165","citation_count":82,"is_preprint":false},{"pmid":"35246532","id":"PMC_35246532","title":"In vitro maturation of Toxoplasma gondii bradyzoites in human myotubes and their metabolomic characterization.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35246532","citation_count":46,"is_preprint":false},{"pmid":"32959958","id":"PMC_32959958","title":"Toxoplasma gondii α-amylase deletion mutant is a promising vaccine against acute and chronic toxoplasmosis.","date":"2020","source":"Microbial biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/32959958","citation_count":27,"is_preprint":false},{"pmid":"36702847","id":"PMC_36702847","title":"Rapid metabolic reprogramming mediated by the AMP-activated protein kinase during the lytic cycle of Toxoplasma gondii.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36702847","citation_count":21,"is_preprint":false},{"pmid":"36439853","id":"PMC_36439853","title":"The determinants regulating Toxoplasma gondii bradyzoite development.","date":"2022","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/36439853","citation_count":16,"is_preprint":false},{"pmid":"38589375","id":"PMC_38589375","title":"Metabolic plasticity, essentiality and therapeutic potential of ribose-5-phosphate synthesis in Toxoplasma gondii.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38589375","citation_count":11,"is_preprint":false},{"pmid":"33655350","id":"PMC_33655350","title":"Lysine crotonylation is widespread on proteins of diverse functions and localizations in Toxoplasma gondii.","date":"2021","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/33655350","citation_count":5,"is_preprint":false},{"pmid":"40492719","id":"PMC_40492719","title":"Dynamics of amylopectin granule accumulation during the course of chronic Toxoplasma infection is linked to intra-cyst bradyzoite replication.","date":"2025","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/40492719","citation_count":4,"is_preprint":false},{"pmid":"39255306","id":"PMC_39255306","title":"PP1 phosphatase controls both daughter cell formation and amylopectin levels in Toxoplasma gondii.","date":"2024","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/39255306","citation_count":4,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11582,"output_tokens":952,"usd":0.024513,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5797,"output_tokens":1886,"usd":0.038067,"stage2_stop_reason":"end_turn"},"total_usd":0.121501,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard","corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"re-read with subject anchor"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"TgGP (glycogen phosphorylase) is required for amylopectin catabolism in Toxoplasma gondii bradyzoites; a stop codon mutant (S25stop) and a phosphorylation-null mutant (S25A) both display amylopectin accumulation, while a phosphorylation-mimetic mutant (S25E) shows minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions, establishing that phosphorylation of Ser25 activates TgGP to drive amylopectin digestion.\",\n      \"method\": \"Site-directed mutagenesis (S25stop, S25A, S25E) combined with CRISPR/genome editing, amylopectin staining, and complementation rescue experiments in T. gondii\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple separation-of-function mutants with complementation rescue, replicated across growth conditions in a single rigorous study\",\n      \"pmids\": [\"28851850\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TgGP acts downstream of or in the same pathway as CDPK2 in regulating amylopectin consumption: knockout of CDPK2 in the phosphorylation-mimetic TgGP S25E background (PruΔcdpk2/TgGPS25E) still showed minimal amylopectin accumulation, whereas Δcdpk2 combined with inactive GP mutants retained amylopectin accumulation, placing TgGP downstream of CDPK2 activity.\",\n      \"method\": \"Genetic epistasis — CDPK2 knockout introduced into TgGP mutant backgrounds, amylopectin phenotype comparison\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis with multiple double-mutant combinations and defined phenotypic readout in a single rigorous study\",\n      \"pmids\": [\"28851850\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Both loss of TgGP activity (S25A inactive mutant) and constitutive TgGP activity (S25E hyperactive mutant) significantly reduced brain cyst production in mice compared to wild-type, and complementation restoring normal amylopectin regulation restored cyst production to control levels, establishing that tight TgGP-mediated regulation of amylopectin is required for efficient cyst formation.\",\n      \"method\": \"Mouse infection model with cyst quantification, complementation experiment\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function and gain-of-function with complementation rescue, defined in vivo phenotypic readout in a single rigorous study\",\n      \"pmids\": [\"28851850\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TgGP (putative glycogen phosphorylase) was identified as a lysine-crotonylated protein in a global Kcr proteome analysis of T. gondii, indicating it bears at least one lysine crotonylation post-translational modification site.\",\n      \"method\": \"LC-MS/MS proteomics with immune-affinity enrichment for crotonylated peptides\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single proteomics catalog study, no functional validation of the modification on TgGP specifically\",\n      \"pmids\": [\"33655350\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgGP (putative glycogen phosphorylase) is the enzyme responsible for amylopectin catabolism in Toxoplasma gondii bradyzoites, regulated by phosphorylation of Ser25 (activated by phosphorylation, inactivated when dephosphorylated), acts downstream of CDPK2 in the amylopectin regulatory pathway, and requires tight activity control — neither constitutive inactivation nor constitutive activation — for efficient production of brain tissue cysts during chronic infection.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TgGP (putative glycogen phosphorylase) is the enzyme responsible for amylopectin catabolism in Toxoplasma gondii bradyzoites, and its activity is governed by phosphorylation of Ser25: a phosphorylation-null mutant (S25A) and a truncation mutant (S25stop) accumulate amylopectin, whereas a phosphomimetic mutant (S25E) shows minimal accumulation, establishing that Ser25 phosphorylation activates TgGP to drive amylopectin digestion [#0]. Tight control of this activity is required for chronic infection, as both loss of activity (S25A) and constitutive activity (S25E) reduce brain cyst production in mice, while complementation restoring normal amylopectin regulation restores cyst formation [#1]. Beyond this phosphoregulated role in amylopectin turnover, no further mechanistic detail about TgGP has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"It was unknown which enzyme degrades the bradyzoite storage polysaccharide amylopectin and how it is controlled; separation-of-function mutants established that TgGP drives amylopectin catabolism and is switched on by Ser25 phosphorylation.\",\n      \"evidence\": \"Site-directed mutagenesis (S25stop, S25A, S25E) with CRISPR editing, amylopectin staining, and complementation rescue in T. gondii Pru\",\n      \"pmids\": [\"28851850\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The kinase and phosphatase acting on Ser25 are not identified in this finding\",\n        \"No direct biochemical demonstration of phosphorylase catalytic activity on amylopectin substrate\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"It was unknown whether the balance of amylopectin turnover matters for chronic infection; gain- and loss-of-function TgGP mutants showed that both constitutive inactivation and constitutive activation impair brain cyst formation, establishing that tight regulation of TgGP is required in vivo.\",\n      \"evidence\": \"Mouse infection model with cyst quantification and complementation rescue in T. gondii Pru\",\n      \"pmids\": [\"28851850\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Does not resolve how amylopectin flux mechanistically supports cyst maturation\",\n        \"Upstream regulatory signals controlling TgGP activity in vivo are not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Whether TgGP carries additional post-translational modifications was unaddressed; a global crotonylome catalog identified TgGP as a lysine-crotonylated protein, raising the possibility of regulation beyond Ser25 phosphorylation.\",\n      \"evidence\": \"LC-MS/MS proteomics with immune-affinity enrichment for crotonylated peptides in T. gondii\",\n      \"pmids\": [\"33655350\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single proteomics catalog with no functional validation of the modification on TgGP\",\n        \"The crotonylated lysine site's effect on TgGP activity is unknown\",\n        \"No enzyme responsible for the modification is identified\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The kinase/phosphatase that sets Ser25 phosphorylation status and the direct catalytic mechanism of TgGP on amylopectin remain uncharacterized in the available corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No identified upstream kinase or phosphatase for Ser25\",\n        \"No in vitro reconstitution of TgGP phosphorylase activity\",\n        \"No structural model of TgGP\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008184\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005980\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": [\n      \"Activity switched on by phosphorylation of Ser25 (S25E phosphomimetic active, S25A phosphonull inactive) [#0]\",\n      \"Tight regulation of TgGP-mediated amylopectin turnover required for efficient brain cyst formation in mice [#1]\",\n      \"Identified as a lysine-crotonylated protein in a global crotonylome catalog (Low confidence, unvalidated) [#2]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_310670","name":"GP","product":"glycogen phosphorylase GP","uniprot_acc":"S8EYF4","chromosome":"TGME49_chrXI","start_pos":1510901},{"strain":"GT1","gene_id":"TGGT1_310670","name":null,"product":"putative glycogen phosphorylase 1","uniprot_acc":null,"chromosome":"TGGT1_chrXI","start_pos":1474522},{"strain":"RH88","gene_id":"TGRH88_050180","name":null,"product":"putative glycogen phosphorylase 1","uniprot_acc":null,"chromosome":"CM023092","start_pos":1560334},{"strain":"VEG","gene_id":"TGVEG_310670","name":null,"product":"putative glycogen phosphorylase 1","uniprot_acc":"A0A125YFU7","chromosome":"TGVEG_chrXI","start_pos":1523084}]}