{"gene":"TG_268590","gene_symbol":"ROM4","run_date":"2026-09-08T14:08:29","timeline":{"discoveries":[{"year":2005,"finding":"TgROM4 is an active intramembrane serine protease (rhomboid) expressed in tachyzoites and localizes to the cell surface/pellicle of T. gondii tachyzoites, where it is uniformly distributed rather than restricted to the posterior pole.","method":"Cloning, expression, cell-based protease activity assay, and immunofluorescence/localization studies","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based activity assay and direct localization by imaging; single lab but two orthogonal methods","pmids":["15753289"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2005,"finding":"TgROM4 has active rhomboid protease activity with similar substrate specificity to other TgROMs, but unlike TgROM5 it was unable to cleave MIC adhesins (e.g., MIC2) in a cell-based assay, indicating substrate specificity differences between ROM4 and ROM5 in this context.","method":"Cell-based protease cleavage assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cell-based functional assay; single lab, single method","pmids":["15753289"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"TgROM4 localizes to the pellicle (plasma membrane) of T. gondii; domain-exchange and site-directed mutagenesis experiments established the topology of TgROM4 at the plasma membrane and identified that N-terminal cytosolic domain determinants govern subcellular sorting of ROM family members.","method":"Domain-exchange experiments, site-directed mutagenesis, immunofluorescence/localization","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — topology established by mutagenesis and domain swaps; single lab","pmids":["18346213"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgROM4 participates in intramembrane proteolytic shedding of surface adhesins including MIC2, AMA1, and MIC3 during gliding motility and invasion. Conditional knockdown of ROM4 causes accumulation of adhesins on the parasite surface, disrupts the apical-posterior adhesin gradient, impairs normal gliding (shifting parasites to posterior twirling), enhances host cell binding but blocks apical orientation and moving junction formation, severely reducing invasion efficiency.","method":"Conditional knockout (tetracycline-regulated knockdown), surface adhesin quantification, gliding motility assay, invasion assay, Western blot for adhesin shedding","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic KO with multiple orthogonal functional readouts (adhesin shedding, gliding, attachment, invasion, moving junction), replicated phenotype consistent across labs","pmids":["20421941"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"ROM4 is the primary rhomboid protease responsible for adhesin shedding (including MIC2) and efficient host cell invasion in T. gondii tachyzoites; ROM1 and ROM5 play negligible roles. Triple knockout of ROM1/ROM4/ROM5 is viable and MIC2 is still partially removed from the surface during invasion, implying ROM-independent adhesin removal mechanisms exist.","method":"Single, double, and triple knockout generation (genetic deletion), adhesin surface accumulation assay, gliding motility assay, invasion assay, moving junction formation assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic knockout combinations with multiple functional readouts; independent confirmation of ROM4 as primary protease, consistent with prior work","pmids":["25336455"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"ROM4 constitutively accounts for the majority of MPP1 (micronemal protein protease 1) activity, cleaving transmembrane MIC substrates MIC2 and MIC6 within their transmembrane segments. In rom4-ko parasites, MIC2 and MIC6 are not shed, and twirling motility predominates with enhanced attachment and impaired invasion, while intracellular growth and egress remain unaffected. ROM5 provides residual cleavage of AMA1 (and other AMA family members) in the absence of ROM4, but ROM5 deletion alone does not alter MIC shedding or invasion.","method":"Cre-recombinase knockout of ROM4, CRISPR-Cas9 knockout of ROM5, substrate cleavage assays, gliding motility assay, invasion assay, egress assay","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent genetic KO strategies (Cre and CRISPR), multiple substrate and functional readouts; independent lab confirming ROM4 primacy","pmids":["25846828"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgROM4 is an active intramembrane serine (rhomboid) protease uniformly distributed on the pellicle/plasma membrane of T. gondii tachyzoites that constitutively mediates the shedding of transmembrane micronemal adhesins (MIC2, MIC6, AMA1, MIC3) during gliding motility and host cell invasion; loss of ROM4 causes adhesin accumulation on the parasite surface, disrupts the apical-to-posterior adhesin gradient, shifts motility from helical to posterior twirling, enhances non-productive host cell attachment, and severely impairs moving junction formation and invasion, while intracellular growth and egress are unaffected, and ROM-independent mechanisms can partially compensate for adhesin removal during entry."},"narrative":{"mechanistic_narrative":"TgROM4 is an active intramembrane serine (rhomboid) protease that constitutively drives the shedding of transmembrane micronemal adhesins during gliding motility and host cell invasion by Toxoplasma gondii tachyzoites [PMID:15753289, PMID:20421941]. Rather than being restricted to the posterior pole, it is uniformly distributed across the pellicle/plasma membrane, a localization governed by N-terminal cytosolic domain determinants that direct sorting of ROM family members [PMID:15753289, PMID:18346213]. ROM4 constitutes the majority of MPP1 activity, cleaving substrates such as MIC2 and MIC6 within their transmembrane segments; conditional knockdown or deletion causes adhesin accumulation on the surface, collapse of the apical-to-posterior adhesin gradient, a shift from helical gliding to posterior twirling, enhanced non-productive attachment, and severe impairment of apical orientation, moving junction formation, and invasion, while intracellular growth and egress are unaffected [PMID:20421941, PMID:25846828]. Genetic dissection establishes ROM4 as the primary invasion-relevant rhomboid, with ROM1 and ROM5 playing negligible roles in MIC shedding; ROM5 provides only residual cleavage of AMA1 in ROM4's absence, and a viable ROM1/ROM4/ROM5 triple knockout that still partially removes surface MIC2 reveals ROM-independent adhesin removal mechanisms [PMID:25336455, PMID:25846828]. In a cell-based assay ROM4 was unable to cleave MIC2, distinguishing its context-dependent substrate handling from ROM5 [PMID:15753289].","teleology":[{"year":2005,"claim":"Establishing that TgROM4 is an enzymatically active rhomboid protease on the parasite surface defined it as a candidate mediator of surface protein processing rather than an inert membrane protein.","evidence":"Cloning, cell-based protease activity assay, and immunofluorescence localization in T. gondii tachyzoites","pmids":["15753289"],"confidence":"Medium","gaps":["Cell-based assay did not identify endogenous substrates","ROM4 failed to cleave MIC2 in this heterologous context, leaving its physiological substrates unresolved","No genetic loss-of-function data at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Determining ROM4 membrane topology and the sequence determinants of its sorting explained how the enzyme is positioned in the pellicle to access surface adhesin substrates.","evidence":"Domain-exchange experiments, site-directed mutagenesis, and localization imaging in T. gondii","pmids":["18346213"],"confidence":"Medium","gaps":["Did not test the functional consequence of mislocalization on invasion","Mechanism linking N-terminal cytosolic determinants to trafficking machinery not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Conditional knockdown demonstrated that ROM4 is functionally required for adhesin shedding, motility, and invasion, converting the biochemical activity into a defined cellular role.","evidence":"Tetracycline-regulated conditional knockdown with adhesin quantification, gliding, attachment, moving junction, and invasion assays in T. gondii","pmids":["20421941"],"confidence":"High","gaps":["Residual protein in conditional knockdown could mask the null phenotype","Whether adhesin accumulation directly causes the twirling/attachment defect versus an indirect effect not fully separated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Systematic single/double/triple rhomboid knockouts established ROM4 as the primary invasion-relevant protease and revealed that ROM-independent mechanisms can partially remove surface adhesins.","evidence":"Genetic deletion of ROM1/ROM4/ROM5 in combination, adhesin accumulation, gliding, moving junction, and invasion assays in T. gondii","pmids":["25336455"],"confidence":"High","gaps":["The ROM-independent adhesin removal mechanism was not identified","Viability of the triple knockout indicates redundancy or non-essentiality not mechanistically explained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Mapping ROM4 as the major MPP1 activity cleaving MIC2/MIC6 within their transmembrane segments, while ROM5 handles residual AMA1 cleavage, resolved the substrate division of labor and confirmed the motility/invasion phenotype with independent genetic strategies.","evidence":"Cre-recombinase and CRISPR-Cas9 knockouts, transmembrane substrate cleavage assays, gliding, invasion, and egress assays in T. gondii","pmids":["25846828"],"confidence":"High","gaps":["Structural basis of transmembrane substrate recognition not determined","How ROM4 activity is spatially and temporally regulated during invasion not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The identity of the ROM-independent adhesin removal machinery and the regulatory control of ROM4 activity during the invasion cycle remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No non-rhomboid protease or shedding mechanism identified for residual MIC2 removal","No structural model of ROM4 substrate engagement","Regulation of ROM4 spatial activity relative to the apical-posterior adhesin gradient unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["MIC2 (TRANSMEMBRANE MICRONEMAL ADHESIN SUBSTRATE)","MIC6 (TRANSMEMBRANE MICRONEMAL ADHESIN SUBSTRATE)","AMA1 (ADHESIN SUBSTRATE)","MIC3 (ADHESIN SUBSTRATE)"],"other_free_text":["ROM4 constitutes the majority of MPP1 (micronemal protein protease 1) activity in tachyzoites [#5]","ROM4 loss shifts gliding from helical to posterior twirling, enhances non-productive attachment, and blocks moving junction formation and invasion without affecting intracellular growth or egress [#3, #5]","ROM1/ROM4/ROM5 triple knockout is viable and MIC2 is still partially shed, indicating ROM-independent adhesin removal mechanisms [#4]","ROM5 provides residual AMA1 cleavage in the absence of ROM4 [#5]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"15753289","id":"PMC_15753289","title":"A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma.","date":"2005","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15753289","citation_count":171,"is_preprint":false},{"pmid":"20421941","id":"PMC_20421941","title":"Rhomboid 4 (ROM4) affects the processing of surface adhesins and facilitates host cell invasion by Toxoplasma gondii.","date":"2010","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/20421941","citation_count":101,"is_preprint":false},{"pmid":"15358257","id":"PMC_15358257","title":"Host cell invasion by the apicomplexans: the significance of microneme protein proteolysis.","date":"2004","source":"Current opinion in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/15358257","citation_count":72,"is_preprint":false},{"pmid":"25336455","id":"PMC_25336455","title":"Functional analysis of rhomboid proteases during Toxoplasma invasion.","date":"2014","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/25336455","citation_count":55,"is_preprint":false},{"pmid":"26111968","id":"PMC_26111968","title":"Protective efficacy of two novel DNA vaccines expressing Toxoplasma gondii rhomboid 4 and rhomboid 5 proteins against acute and chronic toxoplasmosis in mice.","date":"2015","source":"Expert review of vaccines","url":"https://pubmed.ncbi.nlm.nih.gov/26111968","citation_count":41,"is_preprint":false},{"pmid":"25846828","id":"PMC_25846828","title":"Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion.","date":"2015","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/25846828","citation_count":39,"is_preprint":false},{"pmid":"27909791","id":"PMC_27909791","title":"Immunological evaluation of a DNA cocktail vaccine with co-delivery of calcium phosphate nanoparticles (CaPNs) against the Toxoplasma gondii RH strain in BALB/c mice.","date":"2016","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/27909791","citation_count":32,"is_preprint":false},{"pmid":"18346213","id":"PMC_18346213","title":"Identification of trafficking determinants for polytopic rhomboid proteases in Toxoplasma gondii.","date":"2008","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/18346213","citation_count":27,"is_preprint":false},{"pmid":"28077075","id":"PMC_28077075","title":"Protection via a ROM4 DNA vaccine and peptide against Toxoplasma gondii in BALB/c mice.","date":"2017","source":"BMC infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/28077075","citation_count":25,"is_preprint":false},{"pmid":"21718637","id":"PMC_21718637","title":"[TgROM4 and TgAMA1: a checkpoint in parasite replication].","date":"2011","source":"Medecine sciences : M/S","url":"https://pubmed.ncbi.nlm.nih.gov/21718637","citation_count":2,"is_preprint":false},{"pmid":"29317874","id":"PMC_29317874","title":"Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4) of Tachyzoite of Toxoplasma gondii RH Strain.","date":"2017","source":"Iranian journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/29317874","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7543,"output_tokens":1780,"usd":0.024664,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7065,"output_tokens":3029,"usd":0.055525,"stage2_stop_reason":"end_turn"},"total_usd":0.080189,"stage1_batch_id":"msgbatch_015xmQKDRqnTh9anE4m7LiD6","stage2_batch_id":"msgbatch_01JLpkFTVhoQBNgqHCKFrVVU","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"TgROM4 is an active intramembrane serine protease (rhomboid) expressed in tachyzoites and localizes to the cell surface/pellicle of T. gondii tachyzoites, where it is uniformly distributed rather than restricted to the posterior pole.\",\n      \"method\": \"Cloning, expression, cell-based protease activity assay, and immunofluorescence/localization studies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based activity assay and direct localization by imaging; single lab but two orthogonal methods\",\n      \"pmids\": [\"15753289\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TgROM4 has active rhomboid protease activity with similar substrate specificity to other TgROMs, but unlike TgROM5 it was unable to cleave MIC adhesins (e.g., MIC2) in a cell-based assay, indicating substrate specificity differences between ROM4 and ROM5 in this context.\",\n      \"method\": \"Cell-based protease cleavage assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cell-based functional assay; single lab, single method\",\n      \"pmids\": [\"15753289\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TgROM4 localizes to the pellicle (plasma membrane) of T. gondii; domain-exchange and site-directed mutagenesis experiments established the topology of TgROM4 at the plasma membrane and identified that N-terminal cytosolic domain determinants govern subcellular sorting of ROM family members.\",\n      \"method\": \"Domain-exchange experiments, site-directed mutagenesis, immunofluorescence/localization\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — topology established by mutagenesis and domain swaps; single lab\",\n      \"pmids\": [\"18346213\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgROM4 participates in intramembrane proteolytic shedding of surface adhesins including MIC2, AMA1, and MIC3 during gliding motility and invasion. Conditional knockdown of ROM4 causes accumulation of adhesins on the parasite surface, disrupts the apical-posterior adhesin gradient, impairs normal gliding (shifting parasites to posterior twirling), enhances host cell binding but blocks apical orientation and moving junction formation, severely reducing invasion efficiency.\",\n      \"method\": \"Conditional knockout (tetracycline-regulated knockdown), surface adhesin quantification, gliding motility assay, invasion assay, Western blot for adhesin shedding\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic KO with multiple orthogonal functional readouts (adhesin shedding, gliding, attachment, invasion, moving junction), replicated phenotype consistent across labs\",\n      \"pmids\": [\"20421941\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROM4 is the primary rhomboid protease responsible for adhesin shedding (including MIC2) and efficient host cell invasion in T. gondii tachyzoites; ROM1 and ROM5 play negligible roles. Triple knockout of ROM1/ROM4/ROM5 is viable and MIC2 is still partially removed from the surface during invasion, implying ROM-independent adhesin removal mechanisms exist.\",\n      \"method\": \"Single, double, and triple knockout generation (genetic deletion), adhesin surface accumulation assay, gliding motility assay, invasion assay, moving junction formation assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic knockout combinations with multiple functional readouts; independent confirmation of ROM4 as primary protease, consistent with prior work\",\n      \"pmids\": [\"25336455\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ROM4 constitutively accounts for the majority of MPP1 (micronemal protein protease 1) activity, cleaving transmembrane MIC substrates MIC2 and MIC6 within their transmembrane segments. In rom4-ko parasites, MIC2 and MIC6 are not shed, and twirling motility predominates with enhanced attachment and impaired invasion, while intracellular growth and egress remain unaffected. ROM5 provides residual cleavage of AMA1 (and other AMA family members) in the absence of ROM4, but ROM5 deletion alone does not alter MIC shedding or invasion.\",\n      \"method\": \"Cre-recombinase knockout of ROM4, CRISPR-Cas9 knockout of ROM5, substrate cleavage assays, gliding motility assay, invasion assay, egress assay\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent genetic KO strategies (Cre and CRISPR), multiple substrate and functional readouts; independent lab confirming ROM4 primacy\",\n      \"pmids\": [\"25846828\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgROM4 is an active intramembrane serine (rhomboid) protease uniformly distributed on the pellicle/plasma membrane of T. gondii tachyzoites that constitutively mediates the shedding of transmembrane micronemal adhesins (MIC2, MIC6, AMA1, MIC3) during gliding motility and host cell invasion; loss of ROM4 causes adhesin accumulation on the parasite surface, disrupts the apical-to-posterior adhesin gradient, shifts motility from helical to posterior twirling, enhances non-productive host cell attachment, and severely impairs moving junction formation and invasion, while intracellular growth and egress are unaffected, and ROM-independent mechanisms can partially compensate for adhesin removal during entry.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgROM4 is an active intramembrane serine (rhomboid) protease that constitutively drives the shedding of transmembrane micronemal adhesins during gliding motility and host cell invasion by Toxoplasma gondii tachyzoites [#0, #3]. Rather than being restricted to the posterior pole, it is uniformly distributed across the pellicle/plasma membrane, a localization governed by N-terminal cytosolic domain determinants that direct sorting of ROM family members [#0, #2]. ROM4 constitutes the majority of MPP1 activity, cleaving substrates such as MIC2 and MIC6 within their transmembrane segments; conditional knockdown or deletion causes adhesin accumulation on the surface, collapse of the apical-to-posterior adhesin gradient, a shift from helical gliding to posterior twirling, enhanced non-productive attachment, and severe impairment of apical orientation, moving junction formation, and invasion, while intracellular growth and egress are unaffected [#3, #5]. Genetic dissection establishes ROM4 as the primary invasion-relevant rhomboid, with ROM1 and ROM5 playing negligible roles in MIC shedding; ROM5 provides only residual cleavage of AMA1 in ROM4's absence, and a viable ROM1/ROM4/ROM5 triple knockout that still partially removes surface MIC2 reveals ROM-independent adhesin removal mechanisms [#4, #5]. In a cell-based assay ROM4 was unable to cleave MIC2, distinguishing its context-dependent substrate handling from ROM5 [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing that TgROM4 is an enzymatically active rhomboid protease on the parasite surface defined it as a candidate mediator of surface protein processing rather than an inert membrane protein.\",\n      \"evidence\": \"Cloning, cell-based protease activity assay, and immunofluorescence localization in T. gondii tachyzoites\",\n      \"pmids\": [\"15753289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Cell-based assay did not identify endogenous substrates\",\n        \"ROM4 failed to cleave MIC2 in this heterologous context, leaving its physiological substrates unresolved\",\n        \"No genetic loss-of-function data at this stage\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Determining ROM4 membrane topology and the sequence determinants of its sorting explained how the enzyme is positioned in the pellicle to access surface adhesin substrates.\",\n      \"evidence\": \"Domain-exchange experiments, site-directed mutagenesis, and localization imaging in T. gondii\",\n      \"pmids\": [\"18346213\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Did not test the functional consequence of mislocalization on invasion\",\n        \"Mechanism linking N-terminal cytosolic determinants to trafficking machinery not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Conditional knockdown demonstrated that ROM4 is functionally required for adhesin shedding, motility, and invasion, converting the biochemical activity into a defined cellular role.\",\n      \"evidence\": \"Tetracycline-regulated conditional knockdown with adhesin quantification, gliding, attachment, moving junction, and invasion assays in T. gondii\",\n      \"pmids\": [\"20421941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Residual protein in conditional knockdown could mask the null phenotype\",\n        \"Whether adhesin accumulation directly causes the twirling/attachment defect versus an indirect effect not fully separated\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Systematic single/double/triple rhomboid knockouts established ROM4 as the primary invasion-relevant protease and revealed that ROM-independent mechanisms can partially remove surface adhesins.\",\n      \"evidence\": \"Genetic deletion of ROM1/ROM4/ROM5 in combination, adhesin accumulation, gliding, moving junction, and invasion assays in T. gondii\",\n      \"pmids\": [\"25336455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The ROM-independent adhesin removal mechanism was not identified\",\n        \"Viability of the triple knockout indicates redundancy or non-essentiality not mechanistically explained\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapping ROM4 as the major MPP1 activity cleaving MIC2/MIC6 within their transmembrane segments, while ROM5 handles residual AMA1 cleavage, resolved the substrate division of labor and confirmed the motility/invasion phenotype with independent genetic strategies.\",\n      \"evidence\": \"Cre-recombinase and CRISPR-Cas9 knockouts, transmembrane substrate cleavage assays, gliding, invasion, and egress assays in T. gondii\",\n      \"pmids\": [\"25846828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of transmembrane substrate recognition not determined\",\n        \"How ROM4 activity is spatially and temporally regulated during invasion not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the ROM-independent adhesin removal machinery and the regulatory control of ROM4 activity during the invasion cycle remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No non-rhomboid protease or shedding mechanism identified for residual MIC2 removal\",\n        \"No structural model of ROM4 substrate engagement\",\n        \"Regulation of ROM4 spatial activity relative to the apical-posterior adhesin gradient unknown\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004252\", \"supporting_discovery_ids\": [0, 1, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004175\", \"supporting_discovery_ids\": [0, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006508\", \"supporting_discovery_ids\": [3, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0052126\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0071976\", \"supporting_discovery_ids\": [3, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MIC2 (transmembrane micronemal adhesin substrate)\",\n      \"MIC6 (transmembrane micronemal adhesin substrate)\",\n      \"AMA1 (adhesin substrate)\",\n      \"MIC3 (adhesin substrate)\"\n    ],\n    \"other_free_text\": [\n      \"ROM4 constitutes the majority of MPP1 (micronemal protein protease 1) activity in tachyzoites [#5]\",\n      \"ROM4 loss shifts gliding from helical to posterior twirling, enhances non-productive attachment, and blocks moving junction formation and invasion without affecting intracellular growth or egress [#3, #5]\",\n      \"ROM1/ROM4/ROM5 triple knockout is viable and MIC2 is still partially shed, indicating ROM-independent adhesin removal mechanisms [#4]\",\n      \"ROM5 provides residual AMA1 cleavage in the absence of ROM4 [#5]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_268590","name":"ROM4","product":"rhomboid protease ROM4","uniprot_acc":"S8EXP2","chromosome":"TGME49_chrVIII","start_pos":6405734},{"strain":"GT1","gene_id":"TGGT1_268590","name":null,"product":"rhomboid protease ROM4","uniprot_acc":null,"chromosome":"TGGT1_chrVIII","start_pos":6404350},{"strain":"RH88","gene_id":"TGRH88_082680","name":null,"product":"rhomboid protease ROM4","uniprot_acc":null,"chromosome":"CM023089","start_pos":11524439},{"strain":"VEG","gene_id":"TGVEG_268590","name":null,"product":"rhomboid protease ROM4","uniprot_acc":"V4Z806","chromosome":"TGVEG_chrVIII","start_pos":6421417}]}