{"gene":"TG_288380","gene_symbol":"HSP90","run_date":"2026-09-08T14:08:30","timeline":{"discoveries":[{"year":2005,"finding":"TgHsp90 transcript and protein levels increase under stress or bradyzoite differentiation conditions. Fluorescence microscopy revealed that Hsp90 is cytosolic in tachyzoites but present in both nucleus and cytosol in mature bradyzoites, indicating developmentally regulated subcellular redistribution. Geldanamycin (a benzoquinone ansamycin that binds and disrupts Hsp90 function) blocked conversion both from tachyzoite to bradyzoite and bradyzoite to tachyzoite stages, establishing an essential role for Hsp90 in stage interconversion.","method":"Fluorescence microscopy, Western blot, geldanamycin pharmacological inhibition in RH ΔUPRT and ME49/PK strains","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence, pharmacological inhibition with specific phenotypic readout, single lab with multiple orthogonal methods","pmids":["15967463"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH ΔUPRT"},{"species":"Toxoplasma gondii","strain":"ME49"},{"species":"Toxoplasma gondii","strain":"PK"}]},{"year":2003,"finding":"TgHsp90 (82 kDa) is a cytoplasmic protein secreted from extracellular tachyzoites but not released into the parasitophorous vacuole after invasion. Geldanamycin treatment did not affect TgHsp90 secretion from extracellular tachyzoites but significantly disturbed host cell entry and intracellular parasite growth, indicating TgHsp90 function is required for invasion and intracellular replication.","method":"Monoclonal antibody characterization, cDNA cloning and sequencing, geldanamycin pharmacological inhibition, invasion and growth assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional pharmacological inhibition with specific invasion/growth phenotype, supported by molecular cloning and localization data, single lab","pmids":["14623321"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Genetic knockout of TgHsp90 (ΔHsp90) resulted in defective host-cell invasion, failure to proliferate in vitro in Vero cells, and loss of long-term survival in mice in vivo. These defects were rescued by complementation with TgHsp90, establishing that TgHsp90 is essential for invasion, replication, and parasite viability in vivo.","method":"Gene knockout (ΔHsp90), complementation strain, in vitro invasion and proliferation assays, mouse survival model","journal":"Parasite (Paris, France)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with specific phenotypic readouts in multiple contexts (in vitro and in vivo) plus complementation rescue, confirming essentiality","pmids":["28627357"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"TgSis1 (a Hsp40/J-domain protein) co-immunoprecipitated with both TgHsp70 and TgHsp90, indicating that TgHsp90 participates in a cytosolic chaperone complex with TgHsp70 and TgSis1.","method":"Co-immunoprecipitation, immunofluorescence localization","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP demonstrating complex membership, single lab, localization corroborated but no functional mutagenesis","pmids":["22209934"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgUNC (a UCS-family myosin co-chaperone) enables functional expression of TgMyoA (class XIVa myosin) in Sf9 cells; its tetratricopeptide repeat (TPR) domain was not essential for obtaining functional myosin, implying alternative mechanisms exist for Hsp90 recruitment to TgMyoA during folding.","method":"Baculovirus/Sf9 heterologous expression, co-expression with TgUNC, in vitro actin motility assay, domain deletion analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with functional motility assay demonstrating Hsp90 co-chaperone role in myosin folding, single lab","pmids":["25231988"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"Quercetin, a bioflavonoid that inhibits synthesis of Hsp90, Hsp70, and Hsp27, suppresses induction of bradyzoite development in vitro, implicating heat shock proteins including Hsp90 in the process of bradyzoite differentiation.","method":"Quercetin pharmacological inhibition, immunofluorescence with bradyzoite-specific antibodies, Western blot, RT-PCR","journal":"Infection and immunity","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibitor is not specific to Hsp90 alone (also inhibits Hsp70 and Hsp27), single lab, no genetic confirmation","pmids":["9632598"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"Following bag1 (HSP30) knockout in ME49, transcription of HSP90 (along with HSP20, HSP21, HSP40, HSP60, HSP70, and BFD1) was significantly upregulated, indicating that TgHsp90 participates in a compensatory stress-response network that partially rescues bradyzoite differentiation in the absence of BAG1.","method":"Gene knockout (ME49Δbag1), qPCR for HSP family transcript levels, Western blot, cyst formation assay","journal":"Parasites & vectors","confidence":"Low","confidence_rationale":"Tier 3 / Weak — transcriptional upregulation of Hsp90 inferred from qPCR in a bag1 KO background; no direct functional experiment on Hsp90 itself","pmids":["39080770"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2026,"finding":"In a murine ocular toxoplasmosis model, HDAC6 inhibition by Tubastatin A regulated the HDAC6-Hsp90 pathway, leading to decreased VEGF and HSF1 expression. This places TgHsp90 (or the host Hsp90 pathway) downstream of HDAC6 in the context of retinal infection pathology.","method":"Intravitreal injection of Tubastatin A (HDAC6 inhibitor), immunofluorescence, cytokine measurement, histopathology in murine OT model","journal":"ACS infectious diseases","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pathway placement inferred from pharmacological inhibition in a complex in vivo model; Hsp90 involvement is correlative and the experiment does not distinguish host vs. parasite Hsp90","pmids":["41533437"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgHsp90 is an essential, developmentally regulated cytosolic molecular chaperone in Toxoplasma gondii that localizes to the cytosol in tachyzoites and redistributes to both nucleus and cytosol in bradyzoites; it is required for host-cell invasion and intracellular replication (demonstrated by genetic knockout with complementation rescue), participates in a chaperone complex with TgHsp70 and the co-chaperone TgSis1, assists folding of the class XIVa myosin TgMyoA via the UCS co-chaperone TgUNC, and its pharmacological inhibition by geldanamycin blocks tachyzoite-to-bradyzoite and bradyzoite-to-tachyzoite interconversion, indicating an essential role in stage switching."},"narrative":{"mechanistic_narrative":"TgHsp90 is an essential, developmentally regulated cytosolic molecular chaperone in Toxoplasma gondii required for host-cell invasion, intracellular replication, and stage interconversion [PMID:28627357, PMID:15967463]. Genetic knockout abolishes invasion and proliferation in vitro and long-term parasite survival in mice, with defects reversed by complementation, establishing that TgHsp90 is essential for the lytic cycle and in vivo viability [PMID:28627357]. TgHsp90 is a cytoplasmic protein secreted from extracellular tachyzoites, and its pharmacological inhibition by geldanamycin disturbs host-cell entry and intracellular growth without affecting secretion, placing its function at the level of invasion and replication rather than release [PMID:14623321]. Its transcript and protein levels rise under stress and bradyzoite differentiation conditions, and the protein redistributes from a cytosolic pool in tachyzoites to both nucleus and cytosol in mature bradyzoites; geldanamycin blocks both directions of tachyzoite-bradyzoite interconversion, implicating TgHsp90 in developmental stage switching [PMID:15967463]. Mechanistically, TgHsp90 acts within a cytosolic chaperone complex, co-immunoprecipitating with TgHsp70 and the Hsp40/J-domain co-chaperone TgSis1 [PMID:22209934], and contributes to folding of the class XIVa myosin TgMyoA through the UCS co-chaperone TgUNC, whose TPR domain is dispensable for recruitment [PMID:25231988].","teleology":[{"year":2003,"claim":"Establishing whether TgHsp90 activity is needed for the parasite lytic cycle, this work showed the chaperone is a secreted cytoplasmic protein whose function, not its secretion, is required for host entry and replication.","evidence":"Monoclonal antibody characterization, cDNA cloning, and geldanamycin inhibition with invasion and growth assays in T. gondii","pmids":["14623321"],"confidence":"Medium","gaps":["Pharmacological inhibition does not exclude off-target effects","Substrates mediating the invasion/growth requirement not identified","No genetic confirmation of essentiality at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2005,"claim":"Addressing whether TgHsp90 has a role in developmental stage transitions, this study linked its stress-induced upregulation and nuclear/cytosolic redistribution to an essential function in bidirectional tachyzoite-bradyzoite interconversion.","evidence":"Fluorescence microscopy, Western blot, and geldanamycin inhibition in RH ΔUPRT, ME49, and PK strains","pmids":["15967463"],"confidence":"Medium","gaps":["Geldanamycin is not fully Hsp90-specific in vivo","Molecular basis of nuclear redistribution unknown","Client proteins driving stage switching not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH ΔUPRT"},{"species":"Toxoplasma gondii","strain":"ME49"},{"species":"Toxoplasma gondii","strain":"PK"}]},{"year":2011,"claim":"To define the chaperone machinery TgHsp90 operates within, this work placed it in a cytosolic complex with TgHsp70 and the co-chaperone TgSis1.","evidence":"Co-immunoprecipitation and immunofluorescence localization in T. gondii","pmids":["22209934"],"confidence":"Medium","gaps":["Co-IP without reciprocal validation or functional mutagenesis","Stoichiometry and dynamics of the complex unknown","Client specificity of the complex not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Identifying a client and folding route for TgHsp90, this study showed the chaperone contributes to functional TgMyoA folding via the UCS co-chaperone TgUNC, with its TPR domain dispensable, implying alternative Hsp90 recruitment mechanisms.","evidence":"Baculovirus/Sf9 heterologous expression with TgUNC co-expression, in vitro actin motility assay, and domain deletion analysis","pmids":["25231988"],"confidence":"Medium","gaps":["Direct TgHsp90-TgMyoA interaction not demonstrated in the parasite","TPR-independent recruitment mechanism undefined","Other myosin/motility clients not surveyed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Providing definitive genetic proof of essentiality, this work showed that TgHsp90 knockout blocks invasion, proliferation, and in vivo survival, with complementation rescuing all defects.","evidence":"Gene knockout (ΔHsp90), complementation, in vitro invasion/proliferation assays, and a mouse survival model","pmids":["28627357"],"confidence":"High","gaps":["Molecular clients responsible for the phenotypes not mapped","Stage-specific contributions not dissected genetically","Structural determinants of essential function unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"claim":"Early pharmacological evidence implicating heat shock proteins in bradyzoite development showed that quercetin, which inhibits Hsp90/Hsp70/Hsp27 synthesis, suppresses bradyzoite induction.","evidence":"Quercetin inhibition with bradyzoite-specific immunofluorescence, Western blot, and RT-PCR in T. gondii","pmids":["9632598"],"confidence":"Low","gaps":["Inhibitor not specific to Hsp90 (also targets Hsp70 and Hsp27)","No genetic confirmation of Hsp90's specific contribution","Mechanism of differentiation blockade unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Positioning TgHsp90 within a broader stress-response network, this study found HSP90 transcription is upregulated alongside other HSPs and BFD1 in a bag1 knockout background that partially rescues bradyzoite differentiation.","evidence":"ME49Δbag1 knockout with qPCR of HSP-family transcripts, Western blot, and cyst formation assays","pmids":["39080770"],"confidence":"Low","gaps":["Transcriptional readout only, no direct functional test on Hsp90","Compensatory role inferred, not demonstrated","Regulatory link between Hsp90 and BFD1 unestablished"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2026,"claim":"In ocular toxoplasmosis pathology, HDAC6 inhibition modulated the HDAC6-Hsp90 pathway with reduced VEGF and HSF1, placing an Hsp90 pathway downstream of HDAC6 in retinal infection.","evidence":"Intravitreal Tubastatin A (HDAC6 inhibitor) with immunofluorescence, cytokine measurement, and histopathology in a murine ocular toxoplasmosis model","pmids":["41533437"],"confidence":"Low","gaps":["Does not distinguish host from parasite Hsp90","Pathway placement is correlative","Direct Hsp90 involvement not demonstrated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The specific client proteins through which TgHsp90 drives invasion, replication, and stage switching, and the structural/regulatory basis of its nuclear redistribution in bradyzoites, remain undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["Full client repertoire beyond TgMyoA unknown","Mechanism of tachyzoite-to-bradyzoite redistribution uncharacterized","Direct role in stage switching not genetically dissected from chaperone function"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Cytosolic chaperone complex with TgHsp70 and the Hsp40/J-domain co-chaperone TgSis1 [#3]"],"partners":["TGHSP70 [#3]","TGSIS1 (HSP40/J-DOMAIN CO-CHAPERONE) [#3]","TGUNC (UCS-FAMILY MYOSIN CO-CHAPERONE) [#4]","TGMYOA (CLASS XIVA MYOSIN CLIENT) [#4]"],"other_free_text":["Essential for host-cell invasion, intracellular replication, and in vivo viability by genetic knockout with complementation rescue [#2]","Developmentally regulated: upregulated under stress/bradyzoite conditions and required for bidirectional tachyzoite-bradyzoite interconversion, sensitive to geldanamycin [#0]","Secreted from extracellular tachyzoites but not released into the parasitophorous vacuole; secretion is geldanamycin-insensitive whereas invasion/growth are not [#1]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"9632598","id":"PMC_9632598","title":"Bradyzoite development in Toxoplasma gondii and the hsp70 stress response.","date":"1998","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/9632598","citation_count":104,"is_preprint":false},{"pmid":"20955165","id":"PMC_20955165","title":"Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis.","date":"2011","source":"Protein and peptide letters","url":"https://pubmed.ncbi.nlm.nih.gov/20955165","citation_count":90,"is_preprint":false},{"pmid":"15967463","id":"PMC_15967463","title":"Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated.","date":"2005","source":"Journal of molecular 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Drugs and drug resistance","url":"https://pubmed.ncbi.nlm.nih.gov/24533291","citation_count":42,"is_preprint":false},{"pmid":"14623321","id":"PMC_14623321","title":"Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/14623321","citation_count":40,"is_preprint":false},{"pmid":"25231988","id":"PMC_25231988","title":"A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25231988","citation_count":36,"is_preprint":false},{"pmid":"28627357","id":"PMC_28627357","title":"The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth.","date":"2017","source":"Parasite (Paris, France)","url":"https://pubmed.ncbi.nlm.nih.gov/28627357","citation_count":26,"is_preprint":false},{"pmid":"23002958","id":"PMC_23002958","title":"A review of recent patents on the protozoan parasite HSP90 as a drug target.","date":"2013","source":"Recent patents on biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/23002958","citation_count":21,"is_preprint":false},{"pmid":"26528271","id":"PMC_26528271","title":"Heat Shock Protein 90 regulates encystation in Entamoeba.","date":"2015","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/26528271","citation_count":21,"is_preprint":false},{"pmid":"22851753","id":"PMC_22851753","title":"Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection.","date":"2012","source":"Infection and 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  \"finding\": \"TgHsp90 transcript and protein levels increase under stress or bradyzoite differentiation conditions. Fluorescence microscopy revealed that Hsp90 is cytosolic in tachyzoites but present in both nucleus and cytosol in mature bradyzoites, indicating developmentally regulated subcellular redistribution. Geldanamycin (a benzoquinone ansamycin that binds and disrupts Hsp90 function) blocked conversion both from tachyzoite to bradyzoite and bradyzoite to tachyzoite stages, establishing an essential role for Hsp90 in stage interconversion.\",\n      \"method\": \"Fluorescence microscopy, Western blot, geldanamycin pharmacological inhibition in RH ΔUPRT and ME49/PK strains\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence, pharmacological inhibition with specific phenotypic readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"15967463\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"RH ΔUPRT\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"PK\"}\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TgHsp90 (82 kDa) is a cytoplasmic protein secreted from extracellular tachyzoites but not released into the parasitophorous vacuole after invasion. Geldanamycin treatment did not affect TgHsp90 secretion from extracellular tachyzoites but significantly disturbed host cell entry and intracellular parasite growth, indicating TgHsp90 function is required for invasion and intracellular replication.\",\n      \"method\": \"Monoclonal antibody characterization, cDNA cloning and sequencing, geldanamycin pharmacological inhibition, invasion and growth assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional pharmacological inhibition with specific invasion/growth phenotype, supported by molecular cloning and localization data, single lab\",\n      \"pmids\": [\"14623321\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Genetic knockout of TgHsp90 (ΔHsp90) resulted in defective host-cell invasion, failure to proliferate in vitro in Vero cells, and loss of long-term survival in mice in vivo. These defects were rescued by complementation with TgHsp90, establishing that TgHsp90 is essential for invasion, replication, and parasite viability in vivo.\",\n      \"method\": \"Gene knockout (ΔHsp90), complementation strain, in vitro invasion and proliferation assays, mouse survival model\",\n      \"journal\": \"Parasite (Paris, France)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with specific phenotypic readouts in multiple contexts (in vitro and in vivo) plus complementation rescue, confirming essentiality\",\n      \"pmids\": [\"28627357\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TgSis1 (a Hsp40/J-domain protein) co-immunoprecipitated with both TgHsp70 and TgHsp90, indicating that TgHsp90 participates in a cytosolic chaperone complex with TgHsp70 and TgSis1.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence localization\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP demonstrating complex membership, single lab, localization corroborated but no functional mutagenesis\",\n      \"pmids\": [\"22209934\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgUNC (a UCS-family myosin co-chaperone) enables functional expression of TgMyoA (class XIVa myosin) in Sf9 cells; its tetratricopeptide repeat (TPR) domain was not essential for obtaining functional myosin, implying alternative mechanisms exist for Hsp90 recruitment to TgMyoA during folding.\",\n      \"method\": \"Baculovirus/Sf9 heterologous expression, co-expression with TgUNC, in vitro actin motility assay, domain deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with functional motility assay demonstrating Hsp90 co-chaperone role in myosin folding, single lab\",\n      \"pmids\": [\"25231988\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Quercetin, a bioflavonoid that inhibits synthesis of Hsp90, Hsp70, and Hsp27, suppresses induction of bradyzoite development in vitro, implicating heat shock proteins including Hsp90 in the process of bradyzoite differentiation.\",\n      \"method\": \"Quercetin pharmacological inhibition, immunofluorescence with bradyzoite-specific antibodies, Western blot, RT-PCR\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibitor is not specific to Hsp90 alone (also inhibits Hsp70 and Hsp27), single lab, no genetic confirmation\",\n      \"pmids\": [\"9632598\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Following bag1 (HSP30) knockout in ME49, transcription of HSP90 (along with HSP20, HSP21, HSP40, HSP60, HSP70, and BFD1) was significantly upregulated, indicating that TgHsp90 participates in a compensatory stress-response network that partially rescues bradyzoite differentiation in the absence of BAG1.\",\n      \"method\": \"Gene knockout (ME49Δbag1), qPCR for HSP family transcript levels, Western blot, cyst formation assay\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — transcriptional upregulation of Hsp90 inferred from qPCR in a bag1 KO background; no direct functional experiment on Hsp90 itself\",\n      \"pmids\": [\"39080770\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In a murine ocular toxoplasmosis model, HDAC6 inhibition by Tubastatin A regulated the HDAC6-Hsp90 pathway, leading to decreased VEGF and HSF1 expression. This places TgHsp90 (or the host Hsp90 pathway) downstream of HDAC6 in the context of retinal infection pathology.\",\n      \"method\": \"Intravitreal injection of Tubastatin A (HDAC6 inhibitor), immunofluorescence, cytokine measurement, histopathology in murine OT model\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pathway placement inferred from pharmacological inhibition in a complex in vivo model; Hsp90 involvement is correlative and the experiment does not distinguish host vs. parasite Hsp90\",\n      \"pmids\": [\"41533437\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"TgHsp90 is an essential, developmentally regulated cytosolic molecular chaperone in Toxoplasma gondii that localizes to the cytosol in tachyzoites and redistributes to both nucleus and cytosol in bradyzoites; it is required for host-cell invasion and intracellular replication (demonstrated by genetic knockout with complementation rescue), participates in a chaperone complex with TgHsp70 and the co-chaperone TgSis1, assists folding of the class XIVa myosin TgMyoA via the UCS co-chaperone TgUNC, and its pharmacological inhibition by geldanamycin blocks tachyzoite-to-bradyzoite and bradyzoite-to-tachyzoite interconversion, indicating an essential role in stage switching.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgHsp90 is an essential, developmentally regulated cytosolic molecular chaperone in Toxoplasma gondii required for host-cell invasion, intracellular replication, and stage interconversion [#2, #0]. Genetic knockout abolishes invasion and proliferation in vitro and long-term parasite survival in mice, with defects reversed by complementation, establishing that TgHsp90 is essential for the lytic cycle and in vivo viability [#2]. TgHsp90 is a cytoplasmic protein secreted from extracellular tachyzoites, and its pharmacological inhibition by geldanamycin disturbs host-cell entry and intracellular growth without affecting secretion, placing its function at the level of invasion and replication rather than release [#1]. Its transcript and protein levels rise under stress and bradyzoite differentiation conditions, and the protein redistributes from a cytosolic pool in tachyzoites to both nucleus and cytosol in mature bradyzoites; geldanamycin blocks both directions of tachyzoite-bradyzoite interconversion, implicating TgHsp90 in developmental stage switching [#0]. Mechanistically, TgHsp90 acts within a cytosolic chaperone complex, co-immunoprecipitating with TgHsp70 and the Hsp40/J-domain co-chaperone TgSis1 [#3], and contributes to folding of the class XIVa myosin TgMyoA through the UCS co-chaperone TgUNC, whose TPR domain is dispensable for recruitment [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing whether TgHsp90 activity is needed for the parasite lytic cycle, this work showed the chaperone is a secreted cytoplasmic protein whose function, not its secretion, is required for host entry and replication.\",\n      \"evidence\": \"Monoclonal antibody characterization, cDNA cloning, and geldanamycin inhibition with invasion and growth assays in T. gondii\",\n      \"pmids\": [\"14623321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pharmacological inhibition does not exclude off-target effects\", \"Substrates mediating the invasion/growth requirement not identified\", \"No genetic confirmation of essentiality at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Addressing whether TgHsp90 has a role in developmental stage transitions, this study linked its stress-induced upregulation and nuclear/cytosolic redistribution to an essential function in bidirectional tachyzoite-bradyzoite interconversion.\",\n      \"evidence\": \"Fluorescence microscopy, Western blot, and geldanamycin inhibition in RH \\u0394UPRT, ME49, and PK strains\",\n      \"pmids\": [\"15967463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Geldanamycin is not fully Hsp90-specific in vivo\", \"Molecular basis of nuclear redistribution unknown\", \"Client proteins driving stage switching not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH \\u0394UPRT\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"PK\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"To define the chaperone machinery TgHsp90 operates within, this work placed it in a cytosolic complex with TgHsp70 and the co-chaperone TgSis1.\",\n      \"evidence\": \"Co-immunoprecipitation and immunofluorescence localization in T. gondii\",\n      \"pmids\": [\"22209934\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-IP without reciprocal validation or functional mutagenesis\", \"Stoichiometry and dynamics of the complex unknown\", \"Client specificity of the complex not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying a client and folding route for TgHsp90, this study showed the chaperone contributes to functional TgMyoA folding via the UCS co-chaperone TgUNC, with its TPR domain dispensable, implying alternative Hsp90 recruitment mechanisms.\",\n      \"evidence\": \"Baculovirus/Sf9 heterologous expression with TgUNC co-expression, in vitro actin motility assay, and domain deletion analysis\",\n      \"pmids\": [\"25231988\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct TgHsp90-TgMyoA interaction not demonstrated in the parasite\", \"TPR-independent recruitment mechanism undefined\", \"Other myosin/motility clients not surveyed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Providing definitive genetic proof of essentiality, this work showed that TgHsp90 knockout blocks invasion, proliferation, and in vivo survival, with complementation rescuing all defects.\",\n      \"evidence\": \"Gene knockout (\\u0394Hsp90), complementation, in vitro invasion/proliferation assays, and a mouse survival model\",\n      \"pmids\": [\"28627357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular clients responsible for the phenotypes not mapped\", \"Stage-specific contributions not dissected genetically\", \"Structural determinants of essential function unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Early pharmacological evidence implicating heat shock proteins in bradyzoite development showed that quercetin, which inhibits Hsp90/Hsp70/Hsp27 synthesis, suppresses bradyzoite induction.\",\n      \"evidence\": \"Quercetin inhibition with bradyzoite-specific immunofluorescence, Western blot, and RT-PCR in T. gondii\",\n      \"pmids\": [\"9632598\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Inhibitor not specific to Hsp90 (also targets Hsp70 and Hsp27)\", \"No genetic confirmation of Hsp90's specific contribution\", \"Mechanism of differentiation blockade unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Positioning TgHsp90 within a broader stress-response network, this study found HSP90 transcription is upregulated alongside other HSPs and BFD1 in a bag1 knockout background that partially rescues bradyzoite differentiation.\",\n      \"evidence\": \"ME49\\u0394bag1 knockout with qPCR of HSP-family transcripts, Western blot, and cyst formation assays\",\n      \"pmids\": [\"39080770\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Transcriptional readout only, no direct functional test on Hsp90\", \"Compensatory role inferred, not demonstrated\", \"Regulatory link between Hsp90 and BFD1 unestablished\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"In ocular toxoplasmosis pathology, HDAC6 inhibition modulated the HDAC6-Hsp90 pathway with reduced VEGF and HSF1, placing an Hsp90 pathway downstream of HDAC6 in retinal infection.\",\n      \"evidence\": \"Intravitreal Tubastatin A (HDAC6 inhibitor) with immunofluorescence, cytokine measurement, and histopathology in a murine ocular toxoplasmosis model\",\n      \"pmids\": [\"41533437\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Does not distinguish host from parasite Hsp90\", \"Pathway placement is correlative\", \"Direct Hsp90 involvement not demonstrated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The specific client proteins through which TgHsp90 drives invasion, replication, and stage switching, and the structural/regulatory basis of its nuclear redistribution in bradyzoites, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Full client repertoire beyond TgMyoA unknown\", \"Mechanism of tachyzoite-to-bradyzoite redistribution uncharacterized\", \"Direct role in stage switching not genetically dissected from chaperone function\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0051082\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051087\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH \\u0394UPRT\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"PK\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006457\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006986\", \"supporting_discovery_ids\": [0, 5, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]}\n    ],\n    \"complexes\": [\n      \"Cytosolic chaperone complex with TgHsp70 and the Hsp40/J-domain co-chaperone TgSis1 [#3]\"\n    ],\n    \"partners\": [\n      \"TgHsp70 [#3]\",\n      \"TgSis1 (Hsp40/J-domain co-chaperone) [#3]\",\n      \"TgUNC (UCS-family myosin co-chaperone) [#4]\",\n      \"TgMyoA (class XIVa myosin client) [#4]\"\n    ],\n    \"other_free_text\": [\n      \"Essential for host-cell invasion, intracellular replication, and in vivo viability by genetic knockout with complementation rescue [#2]\",\n      \"Developmentally regulated: upregulated under stress/bradyzoite conditions and required for bidirectional tachyzoite-bradyzoite interconversion, sensitive to geldanamycin [#0]\",\n      \"Secreted from extracellular tachyzoites but not released into the parasitophorous vacuole; secretion is geldanamycin-insensitive whereas invasion/growth are not [#1]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_288380","name":"HSP90","product":"heat shock protein HSP90","uniprot_acc":"A0A125YVB7","chromosome":"TGME49_chrIX","start_pos":2483244},{"strain":"GT1","gene_id":"TGGT1_288380","name":null,"product":"heat shock protein HSP90","uniprot_acc":null,"chromosome":"TGGT1_chrIX","start_pos":2294323},{"strain":"RH88","gene_id":"TGRH88_013250","name":null,"product":"heat shock protein HSP90","uniprot_acc":null,"chromosome":"CM023090","start_pos":2549787},{"strain":"VEG","gene_id":"TGVEG_288380","name":null,"product":"heat shock protein HSP90","uniprot_acc":"A0A0F7V6Y1","chromosome":"TGVEG_chrIX","start_pos":2518767}]}