{"gene":"TG_308090","gene_symbol":"ROP5","run_date":"2026-09-10T21:09:19","timeline":{"discoveries":[{"year":2006,"finding":"ROP5 is a member of the ROP2 protein family that is not proteolytically processed during trafficking to rhoptries. It is secreted during host cell invasion and associates with the parasitophorous vacuole membrane (PVM). Differential permeabilization experiments showed ROP5 exposes its C-terminus toward the host cell cytoplasm, representing an inverted topology compared to ROP2 and ROP4.","method":"Differential permeabilization of infected cells, immunofluorescence, Western blot","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional implication, single lab with multiple orthogonal methods","pmids":["16879455"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Deletion of the entire ROP5 locus in virulent type I Toxoplasma gondii caused complete loss of acute virulence in mice, and complementation with ROP5 restored lethality to wild-type levels, establishing ROP5 as indispensable for virulence. The ROP5 locus consists of 4–10 tandem genes encoding pseudokinases that are injected directly into host cells.","method":"Genetic deletion of ROP5 locus, complementation, mouse virulence assay, genome-wide association mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined phenotype, complementation rescue, independently replicated in two concurrent PNAS papers","pmids":["21436047","21586633"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2011,"finding":"Crystal structures of a virulent ROP5 isoform in ATP-bound and unbound states revealed that ROP5 is a pseudokinase that binds ATP in a distorted conformation mediated by unusual magnesium coordination sites. Polymorphisms map to two major surfaces including the activation segment. A conserved non-canonical motif replacing the catalytic Asp is required for full in vivo activity of ROP5.","method":"X-ray crystallography (ATP-bound and apo structures), site-directed mutagenesis, in vivo virulence assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with functional mutagenesis validation, single lab but multiple orthogonal methods","pmids":["21708941"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2012,"finding":"ROP5 directly interacts with immunity-related GTPases (IRGs) and can reduce IRG accumulation on the PVM even in the absence of ROP18. ROP18 can only inhibit IRG accumulation on the PVM of strains that also express virulent ROP5 alleles, indicating ROP5 is required for ROP18 function against IRGs.","method":"Co-immunoprecipitation, genetic deletion of ROP5 and ROP18, IRG recruitment assay by immunofluorescence, mouse virulence assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional genetics plus direct IRG interaction, replicated across multiple strain types","pmids":["22761577"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"},{"species":"Toxoplasma gondii","strain":"type III"}]},{"year":2012,"finding":"ROP5 proteins act as essential co-factors for ROP18 by binding IRG proteins and enforcing an inactive GDP-dependent conformation on the IRG target, preventing GTP-dependent activation and simultaneously exposing target threonines on switch I loop for phosphorylation by ROP18, resulting in permanent IRG inactivation.","method":"Biochemical binding assays, genetic epistasis (ROP5 and ROP18 knockouts), GDP-loading/GTP-exchange assays, IRG phosphorylation assays","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution-level biochemical assay combined with genetic epistasis, mechanistic model supported by multiple orthogonal approaches","pmids":["22802726"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2012,"finding":"ROP5 controls virulence by directly regulating the catalytic activity of the active kinase ROP18 in vitro. Both ROP5 and ROP18 are necessary to avoid IRG recruitment and parasite clearance in IFN-γ-activated macrophages. Clearance of Δrop5 mutants was reversed in macrophages lacking Irgm3, and virulence defect was restored in Irgm3−/− mice, placing ROP5 upstream of IRG-mediated clearance.","method":"In vitro kinase activity assay with recombinant proteins, genetic epistasis with Irgm3−/− mice and macrophages, IRG recruitment immunofluorescence, mouse virulence assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of ROP5 regulation of ROP18 kinase, plus genetic epistasis with defined phenotypic readout","pmids":["23144612"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2013,"finding":"Virulent strains of T. gondii avoided Gbp1 recruitment to the PVM in a strain-dependent manner mediated by ROP18 and the pseudokinase ROP5. Increased Gbp1 recruitment to Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages, a process dependent on the autophagy protein Atg5.","method":"Immunofluorescence (Gbp1 recruitment assay), Δrop5 and Δrop18 knockouts, Gbp1−/− and Atg5-deficient macrophages, mouse virulence assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotype, multiple genetic backgrounds tested, functional readout linked to localization","pmids":["23633952"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2014,"finding":"ROP5 forms complexes with active kinases ROP18 and ROP17 (identified by tandem affinity purification from T. gondii). ROP18 and ROP17 target adjacent essential threonine residues in switch region I of IRGs. The combined activities of ROP17 and ROP18, coordinated by ROP5, contribute to avoidance of IRG recruitment to the vacuole, protecting the parasite from clearance.","method":"Tandem affinity purification/mass spectrometry from T. gondii, biochemical substrate mapping, IRG phosphorylation assays, macrophage clearance assay, mouse virulence assay","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 1 / Strong — TAP-MS complex identification plus in vitro biochemical validation of substrate phosphorylation, multiple orthogonal methods","pmids":["24832449"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2014,"finding":"ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG, producing an allosteric change in IRG structure that prevents IRG oligomerization and alters the orientation of threonines targeted by ROP17 and ROP18. ROP5 proteins are highly specific in the IRG partners they bind, and this binding surface is the most polymorphic region of ROP5.","method":"Binding assays (biochemical), structural/allosteric analysis, mutagenesis, IRG oligomerization assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical demonstration of allosteric mechanism, multiple orthogonal approaches in single rigorous study","pmids":["25118287"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2015,"finding":"GRA7 associates with the PVM by direct binding to ROP5, and is identified as an additional component of the ROP5/ROP18 kinase complex. Absence of GRA7 reduces phosphorylation of Irga6 and correlates with increased vacuolar IRG loading and attenuated virulence.","method":"Co-immunoprecipitation (GRA7–ROP5 direct binding), Irga6 phosphorylation assay, IRG recruitment immunofluorescence, mouse virulence assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated by Co-IP, functional consequence via phosphorylation assay and IRG recruitment, multiple methods","pmids":["26247512"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2016,"finding":"Deletion of ROP5 in type II T. gondii severely reduced cyst burdens (>100-fold reduced virulence), and Δrop5 parasites were less resistant to host IRGs, establishing ROP5 as essential for chronic infection as well as acute virulence.","method":"Genetic deletion of 26 ROPK loci including ROP5, cyst burden measurement, IRG resistance assays, mouse infection models","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with quantified chronic infection phenotype plus mechanistic link to IRG resistance","pmids":["27165797"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2019,"finding":"Wild-derived Eurasian mice resist virulent T. gondii because the IRG protein Irgb2-b1CIM directly binds ROP5 isoform B. The Irgb2-b1 binding interface is highly polymorphic and under positive selection. South American T. gondii strains are virulent in wild-derived mice due to polymorphic ROP5 isoforms not targeted by Irgb2-b1CIM, demonstrating co-adaptation between host IRGs and ROP5.","method":"Direct binding assay (Irgb2-b1 to ROP5 isoform B), genetic analysis of wild-derived mouse resistance, mouse virulence assay with multiple T. gondii strains, population genetics","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated biochemically, with genetic validation in multiple host and parasite strain backgrounds","pmids":["30874554"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I RH"},{"species":"Toxoplasma gondii","strain":"South American strains"}]},{"year":2020,"finding":"ROP5 suppresses CD8 T cell IFNγ responses to parasite-infected macrophages. Multiple ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A/D strains, suppress CD8 T cell IFNγ responses independently of parasite virulence or known IRG–ROP5 interactions. Host NLRP3 (inflammasome-independent) and NLRP1 pathways, not ROP5 removal alone, are absolute requirements for maximal T cell IFNγ production.","method":"ROP5 knockout parasites, multiple ROP5 isoform/allele variants, transnuclear CD8 T cell (T57) assay with BMDMs, NLRP3/NLRP1-deficient macrophages","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO plus multiple isoform variants, single lab, defined cellular readout","pmids":["32853276"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"In vivo CRISPR screen in C57BL/6 mice identified ROP5 as an IFN-γ-dependent virulence gene. Combined usage of Ifngr1−/− mice as a comparator highlighted ROP5 (along with ROP18, GRA12, GRA45) as genes whose virulence function is specifically dependent on the IFN-γ response.","method":"In vivo CRISPR screen in virulent T. gondii-infected mice, comparative analysis with Ifngr1−/− mice","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo functional screen with host genetic comparator, defines IFN-γ dependence of ROP5 virulence function","pmids":["37269286"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2024,"finding":"ROP5 interacts with STING through its predicted domain 2 and enhances K63-linked ubiquitination of STING in a predicted domain 3-dependent manner, thereby promoting cGAS-STING-mediated type I IFN responses. ROP5-deficient PRU parasites (PRUΔROP5) induced fewer type I IFN-related immune responses and replicated faster in RAW264.7 macrophages.","method":"Co-immunoprecipitation (ROP5–STING interaction), ubiquitination assay (K63-linked), domain deletion/mutagenesis, ROP5 knockout parasite replication assay","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct Co-IP plus ubiquitination assay with domain mapping, single lab, KO validation","pmids":["39457045"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"PRU (type II)"}]},{"year":2025,"finding":"Among the three major ROP5 isoforms (A, B, C), ROP5B is primarily responsible for the heightened virulence of type I T. gondii in laboratory mice. In vivo virulence correlates with diminished vacuolar IRG protein loading and parasite control in vitro only in the presence of ROP5B but not ROP5A or ROP5C. ROP5A and ROP5C may have co-evolved with IRG proteins in other intermediate host species.","method":"Reverse genetics — expression of individual ROP5 isoforms (A, B, C) in RHΔrop5 background, mouse virulence assay, IRG recruitment immunofluorescence, in vitro macrophage clearance assay","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic isoform dissection by reverse genetics with defined in vivo and in vitro phenotypes, multiple orthogonal readouts","pmids":["40703524"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I RH"}]},{"year":2025,"finding":"Cysteine S-nitrosylation (SNO) of ROP5 paralogs occurs in a host iNOS-dependent manner. RNS (reactive nitrogen species) caused loss of ROP5 association with the PVM, which is necessary for known ROP5 functions. ROP5 was required for Type I and Type II parasites to evade IFN-γ-mediated immune clearance in iNOS-deficient macrophages, and infection with ROP5 knockout parasites rescued susceptibility of iNOS-deficient mice to Type II T. gondii.","method":"SNO proteomics (iNOS-dependent), PVM association assay (immunofluorescence) ± RNS, ROP5 knockout in Type I and II parasites, macrophage clearance assay, iNOS−/− mouse infection","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct PTM identification plus functional consequence at PVM, KO rescue in vivo, preprint not yet peer-reviewed","pmids":["41427312"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]}],"current_model":"ROP5 is a pseudokinase secreted from rhoptries into the host cell during invasion, where it associates with the parasitophorous vacuole membrane (PVM) with its C-terminus exposed to the host cytoplasm; it functions as a central scaffold that (i) directly binds IRG proteins and locks them in an inactive GDP-bound conformation to prevent oligomerization and expose phospho-acceptor threonines, (ii) recruits and activates the kinases ROP18 and ROP17 in a multiprotein complex (also containing GRA7) to phosphorylate and permanently inactivate IRGs, thereby blocking IRG/GBP-mediated PVM disruption and parasite clearance; among its isoforms, ROP5B is the principal virulence determinant in laboratory mice; additionally, ROP5 promotes K63-linked ubiquitination of STING to enhance cGAS-STING-type I IFN signaling, and its PVM association—and thus all known functions—is disrupted by host iNOS-dependent cysteine S-nitrosylation."},"narrative":{"mechanistic_narrative":"ROP5 is a polymorphic, tandemly-encoded rhoptry pseudokinase secreted into the host cell during Toxoplasma gondii invasion, where it functions as the central scaffold of a parasitophorous vacuole membrane (PVM)-associated kinase complex that neutralizes the IFN-γ-driven cell-autonomous immune response and is indispensable for both acute virulence and chronic infection [PMID:21436047, PMID:21586633, PMID:27165797, PMID:37269286]. It associates with the PVM with its C-terminus exposed to the host cytoplasm and adopts a distorted ATP-binding pseudokinase fold in which a non-canonical motif replaces the catalytic aspartate [PMID:16879455, PMID:21708941]. Mechanistically, ROP5 directly binds immunity-related GTPases (IRGs) through its most polymorphic surface, allosterically locking them in an inactive GDP-bound state, blocking oligomerization, and reorienting switch-region I threonines for phosphorylation [PMID:22761577, PMID:22802726, PMID:25118287]; it simultaneously binds and activates the kinases ROP18 and ROP17—together with GRA7—so that these threonines are permanently phosphorylated, preventing IRG and GBP recruitment to the PVM and Atg5-dependent parasite clearance [PMID:23144612, PMID:23633952, PMID:24832449, PMID:26247512]. Among its isoforms, ROP5B is the principal virulence determinant in laboratory mice, and its target specificity underlies host-parasite co-adaptation, with wild-derived mouse resistance conferred by an IRG (Irgb2-b1) that binds ROP5 isoform B [PMID:30874554, PMID:40703524]. Beyond IRG antagonism, ROP5 interacts with STING and promotes its K63-linked ubiquitination to enhance cGAS-STING type I IFN signaling [PMID:39457045], and it modulates CD8 T cell IFNγ responses [PMID:32853276]. Its PVM association, and thus its downstream functions, is disrupted by host iNOS-dependent cysteine S-nitrosylation [PMID:41427312].","teleology":[{"year":2006,"claim":"Establishing where ROP5 acts required defining its trafficking and topology, showing it reaches the host-facing PVM rather than remaining an intra-parasite protein.","evidence":"Differential permeabilization, immunofluorescence and Western blot in infected cells","pmids":["16879455"],"confidence":"Medium","gaps":["Molecular function at the PVM not yet defined","Mechanism anchoring the C-terminus to the host-cytoplasm face unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Genetic deletion of the entire ROP5 locus resolved whether ROP5 is functionally important, establishing it as indispensable for acute virulence.","evidence":"ROP5 locus deletion with complementation rescue and mouse virulence assay in type I and II strains","pmids":["21436047","21586633"],"confidence":"High","gaps":["Which host pathway ROP5 antagonizes not yet identified","Contribution of individual tandem copies unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2011,"claim":"Crystal structures answered whether ROP5 is a catalytically active kinase, revealing it as a pseudokinase that binds ATP in a distorted conformation and mapping polymorphisms to functional surfaces.","evidence":"X-ray crystallography (ATP-bound and apo), site-directed mutagenesis, in vivo virulence assay in type I","pmids":["21708941"],"confidence":"High","gaps":["Binding partners engaged by the polymorphic surfaces not yet defined","Functional role of ATP binding unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2012,"claim":"Multiple studies established the core mechanism: ROP5 directly binds IRGs, is required for ROP18 function, and enforces an inactive GDP-bound IRG conformation that exposes phospho-acceptor threonines for permanent inactivation.","evidence":"Co-IP, genetic epistasis (rop5/rop18 knockouts), GDP/GTP-exchange and IRG phosphorylation assays, in vitro kinase assays, Irgm3-/- mice and macrophages","pmids":["22761577","22802726","23144612"],"confidence":"High","gaps":["Structural basis of the allosteric change not yet resolved","Composition of the full PVM complex incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"},{"species":"Toxoplasma gondii","strain":"type III"}]},{"year":2013,"claim":"Broadening the effector target and testing cross-species conservation showed ROP5 also blocks Gbp1 recruitment/Atg5-dependent clearance and that Hammondia hammondi orthologs are functional in T. gondii.","evidence":"Gbp1 recruitment immunofluorescence with Gbp1-/- and Atg5-deficient macrophages; heterologous complementation with HhROP5 and mouse virulence assay","pmids":["23633952","23589877"],"confidence":"High","gaps":["Whether GBP antagonism is direct or downstream of IRG control unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2014,"claim":"Complex composition and the structural basis of IRG inactivation were resolved: ROP5 scaffolds ROP18 and ROP17 which phosphorylate adjacent switch-I threonines, and ROP5's polymorphic surface binds the IRG nucleotide-binding domain to block oligomerization allosterically.","evidence":"TAP-MS complex identification, biochemical substrate mapping, allosteric/oligomerization binding assays, mutagenesis, macrophage clearance and mouse virulence assays in type I","pmids":["24832449","25118287"],"confidence":"High","gaps":["High-resolution ROP5-IRG co-structure not reported","Determinants of ROP5 isoform-specific IRG partner selection incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2015,"claim":"The complex and virulence role were extended: GRA7 was identified as a direct ROP5-binding PVM component required for efficient IRG phosphorylation, and CRISPR deletion across divergent South American strains confirmed ROP5 as a broadly conserved virulence determinant genetically interacting with ROP18.","evidence":"Co-IP (GRA7-ROP5), Irga6 phosphorylation and IRG recruitment assays; CRISPR-Cas9 knockout across type 10/8/4 strains and two-locus QTL scan","pmids":["26247512","26291965"],"confidence":"High","gaps":["Stoichiometry and assembly order of the ROP5/ROP18/ROP17/GRA7 complex unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type 10 VAND"},{"species":"Toxoplasma gondii","strain":"type 8 TgCtBr5"},{"species":"Toxoplasma gondii","strain":"type 4 TgCtBr18"}]},{"year":2016,"claim":"Deletion in type II parasites showed ROP5 is essential for chronic as well as acute infection, extending its role beyond acute lethality.","evidence":"Deletion of 26 ROPK loci including ROP5, cyst burden measurement and IRG resistance assays in mouse models (type II)","pmids":["27165797"],"confidence":"High","gaps":["Mechanism of ROP5 action during chronic-stage infection not dissected"],"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2019,"claim":"Host-parasite co-adaptation was demonstrated: wild-derived mouse resistance is conferred by an IRG (Irgb2-b1) that directly binds ROP5 isoform B, and South American strains escape via polymorphic ROP5 isoforms.","evidence":"Direct binding assay (Irgb2-b1 to ROP5B), genetic analysis of wild-derived mouse resistance, mouse virulence assays and population genetics","pmids":["30874554"],"confidence":"High","gaps":["Full repertoire of IRG partners for each ROP5 isoform not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I RH"},{"species":"Toxoplasma gondii","strain":"South American strains"}]},{"year":2020,"claim":"Additional immune-modulatory roles emerged: ROP5 suppresses CD8 T cell IFNγ responses independently of virulence/IRG interactions, with maximal responses requiring host NLRP3/NLRP1.","evidence":"ROP5 knockout and multiple isoform variants tested in transnuclear CD8 T cell (T57) assays with NLRP3/NLRP1-deficient BMDMs","pmids":["32853276"],"confidence":"Medium","gaps":["Molecular target through which ROP5 suppresses T cell IFNγ not identified","Relationship to inflammasome sensors unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"An in vivo CRISPR screen with Ifngr1-/- comparators established that ROP5 virulence is specifically IFN-γ-dependent, consolidating its role as a counter-immune effector.","evidence":"In vivo CRISPR screen in virulent T. gondii-infected C57BL/6 vs Ifngr1-/- mice (type I)","pmids":["37269286"],"confidence":"Medium","gaps":["Screen does not add molecular mechanism beyond confirming IFN-γ dependence"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2024,"claim":"A distinct signaling role was defined: ROP5 binds STING via predicted domain 2 and enhances its K63-linked ubiquitination (domain 3-dependent) to promote cGAS-STING type I IFN responses.","evidence":"Co-IP (ROP5-STING), K63-linked ubiquitination assay, domain deletion/mutagenesis, ROP5 knockout PRU replication assay in RAW264.7 macrophages","pmids":["39457045"],"confidence":"Medium","gaps":["How enhancing type I IFN benefits the parasite is not reconciled with its IRG-antagonist role","Single-lab Co-IP without reciprocal structural validation"],"attribution":[{"species":"Toxoplasma gondii","strain":"PRU (type II)"}]},{"year":2025,"claim":"Systematic isoform dissection established ROP5B as the principal virulence determinant in laboratory mice, tying isoform-specific IRG antagonism to the phenotype.","evidence":"Reverse genetics expressing individual ROP5 isoforms (A, B, C) in RHΔrop5, mouse virulence, IRG recruitment and macrophage clearance assays (type I RH)","pmids":["40703524"],"confidence":"High","gaps":["Host-species targets of ROP5A and ROP5C not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I RH"}]},{"year":2025,"claim":"A host countermeasure was identified: iNOS-dependent cysteine S-nitrosylation of ROP5 strips it from the PVM, disabling its functions and rescuing susceptibility of iNOS-deficient mice.","evidence":"SNO proteomics, PVM association assay ± RNS, ROP5 knockout in type I and II, macrophage clearance and iNOS-/- mouse infection (preprint)","pmids":["41427312"],"confidence":"Medium","gaps":["Specific nitrosylated cysteines not fully mapped","Preprint not yet peer-reviewed"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["ROP5/ROP18/ROP17 kinase complex (TAP-MS identified) [#8]","ROP5-GRA7-containing PVM kinase complex [#12]"],"partners":["IRG PROTEINS (IMMUNITY-RELATED GTPASES; E.G. IRGA6) [#3, #4, #9]","IRGB2-B1 (HOST IRG, ISOFORM-SPECIFIC BINDING TO ROP5B) [#14]","ROP18 (ACTIVE KINASE) [#3, #5, #8]","ROP17 (ACTIVE KINASE) [#8]","GRA7 (DIRECT BINDING) [#12]","STING (VIA PREDICTED DOMAIN 2) [#20]"],"other_free_text":["Pseudokinase with distorted ATP binding and a non-canonical motif replacing the catalytic aspartate [#2]","Encoded by a tandem-repeat locus of multiple gene copies with copy-number variation [#1, #17]","Multiple isoforms (A, B, C); ROP5B is the principal virulence determinant in laboratory mice [#21]","PVM association disrupted by host iNOS-dependent cysteine S-nitrosylation [#22]","Rhoptry sorting affected by GGT-2 depletion (ROP5 used as marker) [#18]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"21436047","id":"PMC_21436047","title":"Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21436047","citation_count":205,"is_preprint":false},{"pmid":"24832449","id":"PMC_24832449","title":"The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice.","date":"2014","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/24832449","citation_count":202,"is_preprint":false},{"pmid":"21586633","id":"PMC_21586633","title":"Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21586633","citation_count":184,"is_preprint":false},{"pmid":"22761577","id":"PMC_22761577","title":"The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferon-gamma response.","date":"2012","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/22761577","citation_count":177,"is_preprint":false},{"pmid":"23633952","id":"PMC_23633952","title":"Guanylate-binding protein 1 (Gbp1) contributes to cell-autonomous immunity against Toxoplasma gondii.","date":"2013","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/23633952","citation_count":155,"is_preprint":false},{"pmid":"23144612","id":"PMC_23144612","title":"The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18.","date":"2012","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/23144612","citation_count":139,"is_preprint":false},{"pmid":"22802726","id":"PMC_22802726","title":"A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins.","date":"2012","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/22802726","citation_count":116,"is_preprint":false},{"pmid":"26699401","id":"PMC_26699401","title":"The ROP18 and ROP5 gene 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diseases","url":"https://pubmed.ncbi.nlm.nih.gov/26672224","citation_count":0,"is_preprint":false},{"pmid":"42243401","id":"PMC_42243401","title":"Characterization of ovine abortion in Uruguay reveals extensive non-clonal diversity and multiple evolutionary origins of Toxoplasma gondii.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/42243401","citation_count":0,"is_preprint":false},{"pmid":"26541043","id":"PMC_26541043","title":"[Research Advance on Toxoplasma gondii Rhoptry Protein 2 Family as Candidate Vaccine Molecules].","date":"2015","source":"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/26541043","citation_count":0,"is_preprint":false},{"pmid":"41675383","id":"PMC_41675383","title":"First report of viable Toxoplasma gondii isolation from African lions (Panthera leo): Evidence for their status as intermediate hosts.","date":"2026","source":"Food and waterborne parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/41675383","citation_count":0,"is_preprint":false},{"pmid":"41975467","id":"PMC_41975467","title":"Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system.","date":"2026","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/41975467","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":51808,"output_tokens":6628,"usd":0.127422,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14078,"output_tokens":5696,"usd":0.106395,"stage2_stop_reason":"end_turn"},"total_usd":0.262469,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard","skipped":true,"corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"re-read with subject anchor"},"stage1_raw":"Looking at the papers, I need to identify which ones specifically characterize TGME49_308090 (ROP5) mechanistically, vs. papers that merely mention ROP5 in genotyping/virulence prediction contexts.\n\nThe query asks for findings only if the paper identifies this exact gene. Given that TGME49_308090 is the annotated ROP5 gene in the ME49 strain, and papers [1]-[9], [14]-[17], [23]-[24], [30], [42], [56], [63], [69], [71], [72], [81], [88], [92] describe direct mechanistic experiments on ROP5 protein (localization, complex formation, substrate interaction, structure, etc.), these qualify. Papers that only use ROP5 allele typing for epidemiology/genotyping do NOT qualify.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"ROP5 is a member of the ROP2 protein family that is not proteolytically processed during trafficking to rhoptries. It is secreted during host cell invasion and associates with the parasitophorous vacuole membrane (PVM). Differential permeabilization experiments showed ROP5 exposes its C-terminus toward the host cell cytoplasm, representing an inverted topology compared to ROP2 and ROP4.\",\n      \"method\": \"Differential permeabilization of infected cells, immunofluorescence, Western blot\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional implication, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16879455\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Deletion of the entire ROP5 locus in virulent type I Toxoplasma gondii caused complete loss of acute virulence in mice, and complementation with ROP5 restored lethality to wild-type levels, establishing ROP5 as indispensable for virulence. The ROP5 locus consists of 4–10 tandem genes encoding pseudokinases that are injected directly into host cells.\",\n      \"method\": \"Genetic deletion of ROP5 locus, complementation, mouse virulence assay, genome-wide association mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined phenotype, complementation rescue, independently replicated in two concurrent PNAS papers\",\n      \"pmids\": [\"21436047\", \"21586633\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structures of a virulent ROP5 isoform in ATP-bound and unbound states revealed that ROP5 is a pseudokinase that binds ATP in a distorted conformation mediated by unusual magnesium coordination sites. Polymorphisms map to two major surfaces including the activation segment. A conserved non-canonical motif replacing the catalytic Asp is required for full in vivo activity of ROP5.\",\n      \"method\": \"X-ray crystallography (ATP-bound and apo structures), site-directed mutagenesis, in vivo virulence assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with functional mutagenesis validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21708941\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROP5 directly interacts with immunity-related GTPases (IRGs) and can reduce IRG accumulation on the PVM even in the absence of ROP18. ROP18 can only inhibit IRG accumulation on the PVM of strains that also express virulent ROP5 alleles, indicating ROP5 is required for ROP18 function against IRGs.\",\n      \"method\": \"Co-immunoprecipitation, genetic deletion of ROP5 and ROP18, IRG recruitment assay by immunofluorescence, mouse virulence assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional genetics plus direct IRG interaction, replicated across multiple strain types\",\n      \"pmids\": [\"22761577\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type III\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROP5 proteins act as essential co-factors for ROP18 by binding IRG proteins and enforcing an inactive GDP-dependent conformation on the IRG target, preventing GTP-dependent activation and simultaneously exposing target threonines on switch I loop for phosphorylation by ROP18, resulting in permanent IRG inactivation.\",\n      \"method\": \"Biochemical binding assays, genetic epistasis (ROP5 and ROP18 knockouts), GDP-loading/GTP-exchange assays, IRG phosphorylation assays\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution-level biochemical assay combined with genetic epistasis, mechanistic model supported by multiple orthogonal approaches\",\n      \"pmids\": [\"22802726\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROP5 controls virulence by directly regulating the catalytic activity of the active kinase ROP18 in vitro. Both ROP5 and ROP18 are necessary to avoid IRG recruitment and parasite clearance in IFN-γ-activated macrophages. Clearance of Δrop5 mutants was reversed in macrophages lacking Irgm3, and virulence defect was restored in Irgm3−/− mice, placing ROP5 upstream of IRG-mediated clearance.\",\n      \"method\": \"In vitro kinase activity assay with recombinant proteins, genetic epistasis with Irgm3−/− mice and macrophages, IRG recruitment immunofluorescence, mouse virulence assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of ROP5 regulation of ROP18 kinase, plus genetic epistasis with defined phenotypic readout\",\n      \"pmids\": [\"23144612\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ROP5 and ROP18 virulence function is conserved across species: Hammondia hammondi orthologs of ROP5 (HhROP5) are functional when expressed in T. gondii, with two distinct HhROP5 paralogs increasing the virulence of a T. gondii ROP5 knockout strain.\",\n      \"method\": \"Heterologous complementation of T. gondii Δrop5 with H. hammondi ROP5, mouse virulence 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 — functional complementation across species, single lab\",\n      \"pmids\": [\"23589877\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Virulent strains of T. gondii avoided Gbp1 recruitment to the PVM in a strain-dependent manner mediated by ROP18 and the pseudokinase ROP5. Increased Gbp1 recruitment to Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages, a process dependent on the autophagy protein Atg5.\",\n      \"method\": \"Immunofluorescence (Gbp1 recruitment assay), Δrop5 and Δrop18 knockouts, Gbp1−/− and Atg5-deficient macrophages, mouse virulence assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotype, multiple genetic backgrounds tested, functional readout linked to localization\",\n      \"pmids\": [\"23633952\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROP5 forms complexes with active kinases ROP18 and ROP17 (identified by tandem affinity purification from T. gondii). ROP18 and ROP17 target adjacent essential threonine residues in switch region I of IRGs. The combined activities of ROP17 and ROP18, coordinated by ROP5, contribute to avoidance of IRG recruitment to the vacuole, protecting the parasite from clearance.\",\n      \"method\": \"Tandem affinity purification/mass spectrometry from T. gondii, biochemical substrate mapping, IRG phosphorylation assays, macrophage clearance assay, mouse virulence assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — TAP-MS complex identification plus in vitro biochemical validation of substrate phosphorylation, multiple orthogonal methods\",\n      \"pmids\": [\"24832449\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG, producing an allosteric change in IRG structure that prevents IRG oligomerization and alters the orientation of threonines targeted by ROP17 and ROP18. ROP5 proteins are highly specific in the IRG partners they bind, and this binding surface is the most polymorphic region of ROP5.\",\n      \"method\": \"Binding assays (biochemical), structural/allosteric analysis, mutagenesis, IRG oligomerization assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical demonstration of allosteric mechanism, multiple orthogonal approaches in single rigorous study\",\n      \"pmids\": [\"25118287\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROP5 is secreted via rhoptries and injected into host cells during invasion. The mode of secretion (rhoptry versus dense granule) affects CD8 T cell response magnitude: targeting the ROP5 antigenic epitope to dense granules enhanced CD8 T cell responses, whereas rhoptry delivery reduced them.\",\n      \"method\": \"Transgenic parasites with epitope retargeted to dense granules or rhoptries, CD8 T cell response assays, parasite load measurement in brain\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct experiment showing secretion route determines antigen presentation, single lab, two orthogonal approaches\",\n      \"pmids\": [\"24857659\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ROP5 and ROP18 together are major virulence factors in genetically divergent South American T. gondii strains. CRISPR-Cas9-mediated deletion of all ROP5 copies in VAND (type 10), TgCtBr5 (type 8), and TgCtBr18 (type 4) strains resulted in complete loss of virulence, establishing ROP5 as a conserved virulence determinant across North and South American strains. A significant genetic interaction between ROP5 and ROP18 was identified by two-locus QTL scan.\",\n      \"method\": \"CRISPR-Cas9 knockout, QTL analysis of genetic cross (ME49 × VAND), mouse virulence assay, multiple South American strain backgrounds\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO replicated across four independent strain backgrounds with defined virulence phenotype\",\n      \"pmids\": [\"26291965\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type 10 VAND\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type 8 TgCtBr5\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type 4 TgCtBr18\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA7 associates with the PVM by direct binding to ROP5, and is identified as an additional component of the ROP5/ROP18 kinase complex. Absence of GRA7 reduces phosphorylation of Irga6 and correlates with increased vacuolar IRG loading and attenuated virulence.\",\n      \"method\": \"Co-immunoprecipitation (GRA7–ROP5 direct binding), Irga6 phosphorylation assay, IRG recruitment immunofluorescence, mouse virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated by Co-IP, functional consequence via phosphorylation assay and IRG recruitment, multiple methods\",\n      \"pmids\": [\"26247512\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Deletion of ROP5 in type II T. gondii severely reduced cyst burdens (>100-fold reduced virulence), and Δrop5 parasites were less resistant to host IRGs, establishing ROP5 as essential for chronic infection as well as acute virulence.\",\n      \"method\": \"Genetic deletion of 26 ROPK loci including ROP5, cyst burden measurement, IRG resistance assays, mouse infection models\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with quantified chronic infection phenotype plus mechanistic link to IRG resistance\",\n      \"pmids\": [\"27165797\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Wild-derived Eurasian mice resist virulent T. gondii because the IRG protein Irgb2-b1CIM directly binds ROP5 isoform B. The Irgb2-b1 binding interface is highly polymorphic and under positive selection. South American T. gondii strains are virulent in wild-derived mice due to polymorphic ROP5 isoforms not targeted by Irgb2-b1CIM, demonstrating co-adaptation between host IRGs and ROP5.\",\n      \"method\": \"Direct binding assay (Irgb2-b1 to ROP5 isoform B), genetic analysis of wild-derived mouse resistance, mouse virulence assay with multiple T. gondii strains, population genetics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated biochemically, with genetic validation in multiple host and parasite strain backgrounds\",\n      \"pmids\": [\"30874554\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"South American strains\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ROP5 suppresses CD8 T cell IFNγ responses to parasite-infected macrophages. Multiple ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A/D strains, suppress CD8 T cell IFNγ responses independently of parasite virulence or known IRG–ROP5 interactions. Host NLRP3 (inflammasome-independent) and NLRP1 pathways, not ROP5 removal alone, are absolute requirements for maximal T cell IFNγ production.\",\n      \"method\": \"ROP5 knockout parasites, multiple ROP5 isoform/allele variants, transnuclear CD8 T cell (T57) assay with BMDMs, NLRP3/NLRP1-deficient macrophages\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO plus multiple isoform variants, single lab, defined cellular readout\",\n      \"pmids\": [\"32853276\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA60, a novel dense granule protein, associates with ROP18 and contributes to IRG resistance in type I parasites. Deletion of GRA60 in RHΔrop5 leads to IRG recruitment comparable to single knockouts, indicating GRA60 and ROP5 function in the same IRG-resistance pathway without additive effect.\",\n      \"method\": \"GRA60 knockout and double knockout (GRA60/ROP5), IRG recruitment immunofluorescence, co-immunoprecipitation (GRA60–ROP18 association), mouse virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus Co-IP, single lab, defined IRG recruitment phenotype\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Third-generation (Oxford Nanopore) sequencing resolved the ROP5 locus structure, revealing it as a tandem repeat region containing multiple gene copies. The near-complete assembly confirmed hundreds of tandem repeats including the ROP5 locus, and identified emerging copy number variants in sexual recombinants.\",\n      \"method\": \"Oxford Nanopore long-read sequencing, de novo genome assembly, Hi-C validation\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct genomic characterization of locus structure, single study with parallel validation\",\n      \"pmids\": [\"33906962\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ROP39 was identified in a complex with ROP5B and contributes to acute virulence. ROP39 directly targets Irgb10 and inhibits its homodimer formation, reducing IRG protein loading onto the PVM. This identifies ROP5B as a scaffold for identifying novel IRG-targeting effectors via its interaction partners.\",\n      \"method\": \"Co-immunoprecipitation (ROP39–ROP5B complex), direct binding assay (ROP39–Irgb10), Irgb10 dimerization assay, IRG loading immunofluorescence, mouse virulence assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of ROP5B complex, direct biochemical binding, functional phenotype, single lab\",\n      \"pmids\": [\"36603017\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Depletion of geranylgeranyl transferase GGT-2 (but not GGT-1 or FT) caused diffuse mislocalization of ROP5 (used as a rhoptry marker) and stronger defects in rhoptry protein sorting than other prenyl transferases, indicating GGT-2 contributes to correct sorting of rhoptry proteins including ROP5.\",\n      \"method\": \"Auxin-inducible degron depletion of GGT-2, fluorescent marker assay for ROP5 localization, CRISPR-Cas9 tagging\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ROP5 used as a marker rather than primary subject; localization phenotype without functional follow-up specific to ROP5\",\n      \"pmids\": [\"37108334\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In vivo CRISPR screen in C57BL/6 mice identified ROP5 as an IFN-γ-dependent virulence gene. Combined usage of Ifngr1−/− mice as a comparator highlighted ROP5 (along with ROP18, GRA12, GRA45) as genes whose virulence function is specifically dependent on the IFN-γ response.\",\n      \"method\": \"In vivo CRISPR screen in virulent T. gondii-infected mice, comparative analysis with Ifngr1−/− mice\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo functional screen with host genetic comparator, defines IFN-γ dependence of ROP5 virulence function\",\n      \"pmids\": [\"37269286\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ROP5 interacts with STING through its predicted domain 2 and enhances K63-linked ubiquitination of STING in a predicted domain 3-dependent manner, thereby promoting cGAS-STING-mediated type I IFN responses. ROP5-deficient PRU parasites (PRUΔROP5) induced fewer type I IFN-related immune responses and replicated faster in RAW264.7 macrophages.\",\n      \"method\": \"Co-immunoprecipitation (ROP5–STING interaction), ubiquitination assay (K63-linked), domain deletion/mutagenesis, ROP5 knockout parasite replication assay\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct Co-IP plus ubiquitination assay with domain mapping, single lab, KO validation\",\n      \"pmids\": [\"39457045\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"PRU (type II)\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Among the three major ROP5 isoforms (A, B, C), ROP5B is primarily responsible for the heightened virulence of type I T. gondii in laboratory mice. In vivo virulence correlates with diminished vacuolar IRG protein loading and parasite control in vitro only in the presence of ROP5B but not ROP5A or ROP5C. ROP5A and ROP5C may have co-evolved with IRG proteins in other intermediate host species.\",\n      \"method\": \"Reverse genetics — expression of individual ROP5 isoforms (A, B, C) in RHΔrop5 background, mouse virulence assay, IRG recruitment immunofluorescence, in vitro macrophage clearance assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic isoform dissection by reverse genetics with defined in vivo and in vitro phenotypes, multiple orthogonal readouts\",\n      \"pmids\": [\"40703524\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cysteine S-nitrosylation (SNO) of ROP5 paralogs occurs in a host iNOS-dependent manner. RNS (reactive nitrogen species) caused loss of ROP5 association with the PVM, which is necessary for known ROP5 functions. ROP5 was required for Type I and Type II parasites to evade IFN-γ-mediated immune clearance in iNOS-deficient macrophages, and infection with ROP5 knockout parasites rescued susceptibility of iNOS-deficient mice to Type II T. gondii.\",\n      \"method\": \"SNO proteomics (iNOS-dependent), PVM association assay (immunofluorescence) ± RNS, ROP5 knockout in Type I and II parasites, macrophage clearance assay, iNOS−/− mouse infection\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct PTM identification plus functional consequence at PVM, KO rescue in vivo, preprint not yet peer-reviewed\",\n      \"pmids\": [\"41427312\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    }\n  ],\n  \"current_model\": \"ROP5 is a pseudokinase secreted from rhoptries into the host cell during invasion, where it associates with the parasitophorous vacuole membrane (PVM) with its C-terminus exposed to the host cytoplasm; it functions as a central scaffold that (i) directly binds IRG proteins and locks them in an inactive GDP-bound conformation to prevent oligomerization and expose phospho-acceptor threonines, (ii) recruits and activates the kinases ROP18 and ROP17 in a multiprotein complex (also containing GRA7) to phosphorylate and permanently inactivate IRGs, thereby blocking IRG/GBP-mediated PVM disruption and parasite clearance; among its isoforms, ROP5B is the principal virulence determinant in laboratory mice; additionally, ROP5 promotes K63-linked ubiquitination of STING to enhance cGAS-STING-type I IFN signaling, and its PVM association—and thus all known functions—is disrupted by host iNOS-dependent cysteine S-nitrosylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ROP5 is a polymorphic, tandemly-encoded rhoptry pseudokinase secreted into the host cell during Toxoplasma gondii invasion, where it functions as the central scaffold of a parasitophorous vacuole membrane (PVM)-associated kinase complex that neutralizes the IFN-\\u03b3-driven cell-autonomous immune response and is indispensable for both acute virulence and chronic infection [#1, #13, #19]. It associates with the PVM with its C-terminus exposed to the host cytoplasm and adopts a distorted ATP-binding pseudokinase fold in which a non-canonical motif replaces the catalytic aspartate [#0, #2]. Mechanistically, ROP5 directly binds immunity-related GTPases (IRGs) through its most polymorphic surface, allosterically locking them in an inactive GDP-bound state, blocking oligomerization, and reorienting switch-region I threonines for phosphorylation [#3, #4, #9]; it simultaneously binds and activates the kinases ROP18 and ROP17\\u2014together with GRA7\\u2014so that these threonines are permanently phosphorylated, preventing IRG and GBP recruitment to the PVM and Atg5-dependent parasite clearance [#5, #7, #8, #12]. Among its isoforms, ROP5B is the principal virulence determinant in laboratory mice, and its target specificity underlies host-parasite co-adaptation, with wild-derived mouse resistance conferred by an IRG (Irgb2-b1) that binds ROP5 isoform B [#14, #21]. Beyond IRG antagonism, ROP5 interacts with STING and promotes its K63-linked ubiquitination to enhance cGAS-STING type I IFN signaling [#20], and it modulates CD8 T cell IFN\\u03b3 responses [#15]. Its PVM association, and thus its downstream functions, is disrupted by host iNOS-dependent cysteine S-nitrosylation [#22].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing where ROP5 acts required defining its trafficking and topology, showing it reaches the host-facing PVM rather than remaining an intra-parasite protein.\",\n      \"evidence\": \"Differential permeabilization, immunofluorescence and Western blot in infected cells\",\n      \"pmids\": [\"16879455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular function at the PVM not yet defined\", \"Mechanism anchoring the C-terminus to the host-cytoplasm face unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic deletion of the entire ROP5 locus resolved whether ROP5 is functionally important, establishing it as indispensable for acute virulence.\",\n      \"evidence\": \"ROP5 locus deletion with complementation rescue and mouse virulence assay in type I and II strains\",\n      \"pmids\": [\"21436047\", \"21586633\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which host pathway ROP5 antagonizes not yet identified\", \"Contribution of individual tandem copies unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Crystal structures answered whether ROP5 is a catalytically active kinase, revealing it as a pseudokinase that binds ATP in a distorted conformation and mapping polymorphisms to functional surfaces.\",\n      \"evidence\": \"X-ray crystallography (ATP-bound and apo), site-directed mutagenesis, in vivo virulence assay in type I\",\n      \"pmids\": [\"21708941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding partners engaged by the polymorphic surfaces not yet defined\", \"Functional role of ATP binding unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Multiple studies established the core mechanism: ROP5 directly binds IRGs, is required for ROP18 function, and enforces an inactive GDP-bound IRG conformation that exposes phospho-acceptor threonines for permanent inactivation.\",\n      \"evidence\": \"Co-IP, genetic epistasis (rop5/rop18 knockouts), GDP/GTP-exchange and IRG phosphorylation assays, in vitro kinase assays, Irgm3-/- mice and macrophages\",\n      \"pmids\": [\"22761577\", \"22802726\", \"23144612\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the allosteric change not yet resolved\", \"Composition of the full PVM complex incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type III\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Broadening the effector target and testing cross-species conservation showed ROP5 also blocks Gbp1 recruitment/Atg5-dependent clearance and that Hammondia hammondi orthologs are functional in T. gondii.\",\n      \"evidence\": \"Gbp1 recruitment immunofluorescence with Gbp1-/- and Atg5-deficient macrophages; heterologous complementation with HhROP5 and mouse virulence assay\",\n      \"pmids\": [\"23633952\", \"23589877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GBP antagonism is direct or downstream of IRG control unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Complex composition and the structural basis of IRG inactivation were resolved: ROP5 scaffolds ROP18 and ROP17 which phosphorylate adjacent switch-I threonines, and ROP5's polymorphic surface binds the IRG nucleotide-binding domain to block oligomerization allosterically.\",\n      \"evidence\": \"TAP-MS complex identification, biochemical substrate mapping, allosteric/oligomerization binding assays, mutagenesis, macrophage clearance and mouse virulence assays in type I\",\n      \"pmids\": [\"24832449\", \"25118287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution ROP5-IRG co-structure not reported\", \"Determinants of ROP5 isoform-specific IRG partner selection incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Antigen-delivery experiments established that the rhoptry secretion route of ROP5 shapes CD8 T cell responses, linking its trafficking to immune recognition.\",\n      \"evidence\": \"Transgenic parasites with the ROP5 epitope retargeted to dense granules vs rhoptries, CD8 T cell assays, brain parasite load\",\n      \"pmids\": [\"24857659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking secretion route to presentation efficiency unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The complex and virulence role were extended: GRA7 was identified as a direct ROP5-binding PVM component required for efficient IRG phosphorylation, and CRISPR deletion across divergent South American strains confirmed ROP5 as a broadly conserved virulence determinant genetically interacting with ROP18.\",\n      \"evidence\": \"Co-IP (GRA7-ROP5), Irga6 phosphorylation and IRG recruitment assays; CRISPR-Cas9 knockout across type 10/8/4 strains and two-locus QTL scan\",\n      \"pmids\": [\"26247512\", \"26291965\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the ROP5/ROP18/ROP17/GRA7 complex unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type 10 VAND\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type 8 TgCtBr5\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type 4 TgCtBr18\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Deletion in type II parasites showed ROP5 is essential for chronic as well as acute infection, extending its role beyond acute lethality.\",\n      \"evidence\": \"Deletion of 26 ROPK loci including ROP5, cyst burden measurement and IRG resistance assays in mouse models (type II)\",\n      \"pmids\": [\"27165797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of ROP5 action during chronic-stage infection not dissected\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Host-parasite co-adaptation was demonstrated: wild-derived mouse resistance is conferred by an IRG (Irgb2-b1) that directly binds ROP5 isoform B, and South American strains escape via polymorphic ROP5 isoforms.\",\n      \"evidence\": \"Direct binding assay (Irgb2-b1 to ROP5B), genetic analysis of wild-derived mouse resistance, mouse virulence assays and population genetics\",\n      \"pmids\": [\"30874554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full repertoire of IRG partners for each ROP5 isoform not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"South American strains\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Additional immune-modulatory roles emerged: ROP5 suppresses CD8 T cell IFN\\u03b3 responses independently of virulence/IRG interactions, with maximal responses requiring host NLRP3/NLRP1.\",\n      \"evidence\": \"ROP5 knockout and multiple isoform variants tested in transnuclear CD8 T cell (T57) assays with NLRP3/NLRP1-deficient BMDMs\",\n      \"pmids\": [\"32853276\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular target through which ROP5 suppresses T cell IFN\\u03b3 not identified\", \"Relationship to inflammasome sensors unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"GRA60 was placed in the same IRG-resistance pathway as ROP5, refining the effector network without evidence of an additive effect.\",\n      \"evidence\": \"GRA60 and GRA60/ROP5 double knockouts, IRG recruitment immunofluorescence, Co-IP (GRA60-ROP18), mouse virulence assay in type I RH\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical link between GRA60 and ROP5 not shown\", \"Precise position of GRA60 in the complex unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Long-read sequencing resolved the ROP5 locus as a tandem-repeat array with copy-number variation, clarifying the genomic architecture underlying its multi-copy expression.\",\n      \"evidence\": \"Oxford Nanopore long-read de novo assembly with Hi-C validation in RH and ME49\",\n      \"pmids\": [\"33906962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of copy-number variants not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"An in vivo CRISPR screen with Ifngr1-/- comparators established that ROP5 virulence is specifically IFN-\\u03b3-dependent, consolidating its role as a counter-immune effector.\",\n      \"evidence\": \"In vivo CRISPR screen in virulent T. gondii-infected C57BL/6 vs Ifngr1-/- mice (type I)\",\n      \"pmids\": [\"37269286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Screen does not add molecular mechanism beyond confirming IFN-\\u03b3 dependence\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Prenyltransferase depletion implicated GGT-2 in correct rhoptry sorting of ROP5, though ROP5 served here mainly as a localization marker.\",\n      \"evidence\": \"Auxin-inducible degron depletion of GGT-2, fluorescent marker assay for ROP5 localization, CRISPR tagging\",\n      \"pmids\": [\"37108334\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"ROP5 used as a marker rather than the primary subject; no ROP5-specific functional follow-up\", \"Whether ROP5 itself is prenylated not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A distinct signaling role was defined: ROP5 binds STING via predicted domain 2 and enhances its K63-linked ubiquitination (domain 3-dependent) to promote cGAS-STING type I IFN responses.\",\n      \"evidence\": \"Co-IP (ROP5-STING), K63-linked ubiquitination assay, domain deletion/mutagenesis, ROP5 knockout PRU replication assay in RAW264.7 macrophages\",\n      \"pmids\": [\"39457045\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How enhancing type I IFN benefits the parasite is not reconciled with its IRG-antagonist role\", \"Single-lab Co-IP without reciprocal structural validation\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"PRU (type II)\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Systematic isoform dissection established ROP5B as the principal virulence determinant in laboratory mice, tying isoform-specific IRG antagonism to the phenotype.\",\n      \"evidence\": \"Reverse genetics expressing individual ROP5 isoforms (A, B, C) in RH\\u0394rop5, mouse virulence, IRG recruitment and macrophage clearance assays (type I RH)\",\n      \"pmids\": [\"40703524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host-species targets of ROP5A and ROP5C not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I RH\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A host countermeasure was identified: iNOS-dependent cysteine S-nitrosylation of ROP5 strips it from the PVM, disabling its functions and rescuing susceptibility of iNOS-deficient mice.\",\n      \"evidence\": \"SNO proteomics, PVM association assay \\u00b1 RNS, ROP5 knockout in type I and II, macrophage clearance and iNOS-/- mouse infection (preprint)\",\n      \"pmids\": [\"41427312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific nitrosylated cysteines not fully mapped\", \"Preprint not yet peer-reviewed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ROP5's roles as an IRG/GBP-antagonizing scaffold and as a STING-ubiquitination enhancer that promotes type I IFN are integrated into a single coherent parasite strategy remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No study reconciles IRG antagonism with pro-type-I-IFN STING activity\", \"Molecular target of CD8 T cell IFN\\u03b3 suppression unknown\", \"High-resolution structure of the assembled ROP5/ROP18/ROP17/GRA7 PVM complex not determined\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004672\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]},\n      {\"term_id\": \"GO:0005524\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]},\n      {\"term_id\": \"GO:0019210\", \"supporting_discovery_ids\": [4, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]},\n      {\"term_id\": \"GO:0019901\", \"supporting_discovery_ids\": [3, 5, 8, 9, 14, 20], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]},\n      {\"term_id\": \"GO:0030234\", \"supporting_discovery_ids\": [5, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020008\", \"supporting_discovery_ids\": [0, 10, 18], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051607\", \"supporting_discovery_ids\": [1, 3, 5, 7, 13, 19], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]},\n      {\"term_id\": \"GO:0140546\", \"supporting_discovery_ids\": [7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]},\n      {\"term_id\": \"GO:0032479\", \"supporting_discovery_ids\": [20], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"PRU (type II)\"}]},\n      {\"term_id\": \"GO:0002376\", \"supporting_discovery_ids\": [15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"ROP5/ROP18/ROP17 kinase complex (TAP-MS identified) [#8]\",\n      \"ROP5-GRA7-containing PVM kinase complex [#12]\"\n    ],\n    \"partners\": [\n      \"IRG proteins (immunity-related GTPases; e.g. Irga6) [#3, #4, #9]\",\n      \"Irgb2-b1 (host IRG, isoform-specific binding to ROP5B) [#14]\",\n      \"ROP18 (active kinase) [#3, #5, #8]\",\n      \"ROP17 (active kinase) [#8]\",\n      \"GRA7 (direct binding) [#12]\",\n      \"STING (via predicted domain 2) [#20]\"\n    ],\n    \"other_free_text\": [\n      \"Pseudokinase with distorted ATP binding and a non-canonical motif replacing the catalytic aspartate [#2]\",\n      \"Encoded by a tandem-repeat locus of multiple gene copies with copy-number variation [#1, #17]\",\n      \"Multiple isoforms (A, B, C); ROP5B is the principal virulence determinant in laboratory mice [#21]\",\n      \"PVM association disrupted by host iNOS-dependent cysteine S-nitrosylation [#22]\",\n      \"Rhoptry sorting affected by GGT-2 depletion (ROP5 used as marker) [#18]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_308090","name":"ROP5","product":"rhoptry protein ROP5","uniprot_acc":"A0A125YQ30","chromosome":"TGME49_chrXII","start_pos":566721},{"strain":"GT1","gene_id":"TGGT1_308090","name":null,"product":"rhoptry protein ROP5","uniprot_acc":null,"chromosome":"AAQM03000031","start_pos":206},{"strain":"RH88","gene_id":"TGRH88_057710","name":null,"product":"rhoptry protein ROP5","uniprot_acc":null,"chromosome":"CM023093","start_pos":562599},{"strain":"VEG","gene_id":"TGVEG_308090","name":null,"product":"rhoptry protein ROP5","uniprot_acc":"V4Z6U4","chromosome":"KI544629","start_pos":263}]}