{"gene":"TG_205250","gene_symbol":"ROP18","run_date":"2026-09-05T22:31:42","timeline":{"discoveries":[{"year":2006,"finding":"ROP18 is a polymorphic serine-threonine kinase secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4–5 logs, and these attributes required kinase activity, establishing ROP18 as a secreted effector kinase essential for acute virulence.","method":"Genetic mapping (QTL), positional cloning, transfection/complementation in type III strain, kinase-dead mutant analysis, mouse virulence assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — complementation in vivo with kinase-dead mutagenesis, replicated across two independent papers (PMID:17170305, PMID:17170306)","pmids":["17170305","17170306"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I); type III"}]},{"year":2007,"finding":"Recombinant ROP18 catalytic domain (aa 243–539) possesses serine-threonine protein kinase activity in vitro and phosphorylates a ~70 kDa tachyzoite substrate. Overexpression of ROP18 in transgenic parasites dramatically increases intra-vacuolar parasite multiplication rate in a kinase-activity-dependent manner. ROP18 localises to rhoptries and re-localises to the parasitophorous vacuole membrane (PVM) upon invasion.","method":"In vitro kinase assay with recombinant protein, transgenic overexpression, kinase-dead mutant, immunofluorescence/subcellular fractionation","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with recombinant catalytic domain plus mutagenesis and live-parasite phenotype in a single rigorous study","pmids":["17305424"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2009,"finding":"X-ray crystal structures of pseudokinases ROP2 and ROP8 provided a template for homology modelling of ROP18. Autophosphorylation of key residues in the N-terminal extension activates ROP18; this phosphorylated N-terminus relieves autoinhibition caused by aliphatic side-chain insertion into the ATP-binding pocket. Mutagenesis and mass spectrometry confirmed this activation mechanism, and activated ROP18 in turn phosphorylates ROP2 and ROP8.","method":"X-ray crystallography, homology modelling, autophosphorylation assay, mutagenesis, mass spectrometry","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis and mass spectrometry in one rigorous study","pmids":["19197235"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"ROP18 binds to and phosphorylates multiple immunity-related GTPases (IRGs) on a conserved motif within switch region I of the GTPase domain. This modification prevents IRG recruitment to the PVM, blocks clearance within IFN-γ-activated macrophages and inflammatory monocytes, and is necessary and sufficient to confer parasite survival and virulence in vivo. Substrate profiling revealed the ROP18 phosphorylation consensus.","method":"Co-immunoprecipitation, in vitro phosphorylation assay, substrate profiling, genetic knockout (Δrop18), IFN-γ-activated macrophage clearance assay, mouse virulence assay","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with substrate profiling, reciprocal interaction, KO phenotype, in vivo validation; replicated in same year by PMID:21203588","pmids":["21147463"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2010,"finding":"The type I virulent allele of ROP18 phosphorylates mouse Irga6 at two threonines in the nucleotide-binding domain (switch I region), biochemically inactivating the GTPase and inhibiting its accumulation at the PVM. This phosphorylation event is strain-specific (type I > type II/III) and correlates with virulence in mice.","method":"In vitro phosphorylation assay, phospho-site mapping, GTPase activity assay, confocal microscopy of IRG recruitment, mouse virulence comparison","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical phosphorylation with site mapping and functional GTPase readout, independent replication of IRG targeting alongside PMID:21147463","pmids":["21203588"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type II; type III"}]},{"year":2011,"finding":"ROP18 targets the host ER-bound transcription factor ATF6β. The N-terminal extension of ROP18 interacts with ATF6β and destabilizes it; ROP18 kinase activity is required for proteasome-dependent degradation of ATF6β, which downregulates CD8 T cell-mediated adaptive immune responses. ATF6β-deficient mice are highly susceptible to ROP18-deficient parasites, confirming this axis in vivo.","method":"Co-immunoprecipitation, overexpression in mammalian cells, ROP18 gene disruption (Δrop18), proteasome inhibitor experiments, ATF6β-KO mice challenge","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, KO parasite phenotype, KO host mouse validation, multiple orthogonal methods in one study","pmids":["21670204"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2011,"finding":"ROP18 (active kinase) and ROP5 (pseudokinase) are both required for avoidance of Gbp1 recruitment to the PVM; virulent strains lacking either ROP18 or ROP5 show increased Gbp1 accumulation and clearance in IFN-γ-activated macrophages. The virulence defect of Δrop18 mutants was reversed in Gbp1−/− cells and mice, demonstrating that ROP18 function is upstream of Gbp1-mediated killing.","method":"Genetic knockouts (Δrop18, Δrop5), IFN-γ-activated macrophage clearance assay, Gbp1−/− mice, confocal microscopy","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO parasites, KO host cells, in vivo KO mice; replicated across labs","pmids":["23633952"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2012,"finding":"The arginine-rich N-terminal domain of ROP18 (upstream of the kinase domain) is required for tethering ROP18 to the cytosolic face of the PVM following secretion. Mutants lacking this domain are secreted but fail to associate with the PVM, cannot phosphorylate Irga6, cannot prevent IRG loading onto the PVM, and are avirulent in mice, demonstrating that PVM targeting is essential for ROP18 function.","method":"N-terminal deletion/mutagenesis, confocal microscopy, in vitro phosphorylation assay (Irga6), mouse virulence assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — domain mutagenesis linked to localization, substrate phosphorylation, and in vivo virulence in a single focused study","pmids":["22906355"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2012,"finding":"ROP18 can only efficiently inhibit IRG accumulation on the PVM in strains that co-express virulent ROP5 alleles. ROP5 can directly interact with one or more IRGs and can reduce IRG coating even in the absence of ROP18. Neither ROP18 nor ROP5 markedly affect survival in IFN-γ-activated human cells (which lack the IRG system), indicating that ROP18/ROP5 evolved specifically to block murine IRGs.","method":"Genetic knockouts and allelic swaps, IRG recruitment assay, human cell infection assay, mouse virulence assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple allelic combinations tested across strains, species comparison (mouse vs. human cells), replicated by multiple labs","pmids":["22761577"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type II; type III"}]},{"year":2012,"finding":"ROP5 proteins (pseudokinases) act as essential co-factors for ROP18 by binding adjacent to the nucleotide-binding domain of IRG targets and inducing an allosteric change: this prevents IRG oligomerization and reorients the two threonine residues in switch I that are targeted for phosphorylation by ROP18, thereby preparing the substrate for phosphorylation by ROP18.","method":"Genetic and biochemical binding assays, structural analysis (ROP5 pseudokinase domain), IRG oligomerization assays, in vitro phosphorylation","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — structural + biochemical + genetic epistasis, replicated across multiple papers (PMID:22802726, PMID:23144612, PMID:25118287)","pmids":["22802726"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}]},{"year":2012,"finding":"ROP5 directly regulates the catalytic activity of ROP18 in vitro. Both ROP5 and ROP18 are necessary to avoid IRG recruitment and clearance in IFN-γ-activated macrophages. The virulence defects of Δrop5 and Δrop18 were fully restored in Irgm3−/− mice, placing ROP18 activity genetically downstream of ROP5 and upstream of Irgm3-dependent IRG function.","method":"In vitro kinase activity assay (ROP18 ± ROP5), genetic knockouts, macrophage clearance assay, Irgm3−/− mice epistasis","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro activity regulation plus genetic epistasis in KO hosts; single lab but multiple orthogonal methods","pmids":["23144612"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2013,"finding":"Crystal structure of the ROP18 kinase domain was determined. The structure is inconsistent with a previously proposed autoinhibitory mechanism. A sucrose molecule bound outside the active-site cleft identifies a second ligand-binding pocket; mutational analysis confirmed that this pocket is important for acute virulence in mice.","method":"X-ray crystallography, mutagenesis of second ligand-binding pocket, mouse virulence assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis with in vivo functional validation in a single study","pmids":["24129568"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2014,"finding":"ROP18 exists in a protein complex with pseudokinases ROP8/ROP2 and the dense granule protein GRA7 at the PVM. GRA7 directly binds the active dimer of Irga6 in a GTP-dependent manner, leading to enhanced IRG polymerization and rapid turnover/disassembly. ROP18 and GRA7 act together (synergistically and in the same pathway) to target IRGs by distinct mechanisms; a combined gra7/rop18 double knockout is avirulent while single mutants are only partially attenuated.","method":"Tandem affinity purification-MS, co-immunoprecipitation, in vitro GTPase/polymerization assay, genetic knockouts (Δgra7, Δrop18, double KO), IRG recruitment assay, mouse virulence assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — AP-MS complex identification confirmed by Co-IP, biochemical IRG assay, and double-KO epistasis; single lab","pmids":["24390541"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2014,"finding":"ROP5 forms physical complexes with the active kinases ROP18 and ROP17 (identified by tandem affinity purification in T. gondii). ROP18 and ROP17 together target adjacent essential threonine residues in switch I of IRGs; combined activities of ROP17 and ROP18 are required to prevent IRG recruitment to the PVM and protect the parasite in IFN-γ-activated macrophages.","method":"Tandem affinity purification-MS in T. gondii, in vitro kinase assay, genetic knockouts, IRG recruitment/macrophage clearance assay","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in-parasite complex purification with MS plus biochemical and genetic validation; single lab, multiple orthogonal methods","pmids":["24832449"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2014,"finding":"ROP18 kinase activity and PVM association are both necessary for resistance to host IRGs in type II T. gondii. Deletion of ROP18 (and ROP5, ROP17) in type II severely reduces cyst burdens and >100-fold reduces virulence, establishing ROP18 as essential for chronic infection and not only acute pathogenesis.","method":"Targeted gene deletion of 26 ROPK loci, kinase-dead mutant, PVM-localization mutant, IRG resistance assay, mouse virulence/cyst burden assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic KO with kinase-dead and localization mutants, in vivo chronic infection readout; single lab but comprehensive","pmids":["27165797"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49 (type II)"}]},{"year":2015,"finding":"GRA7 association with the PVM is mediated by direct binding to ROP5, and GRA7 absence results in reduced phosphorylation of Irga6, increased IRG amounts on the vacuole, and attenuated virulence. Among all IRG proteins, Irga6 is the only specific target of ROP18; similarly, GRA7 is strictly an Irga6-specific virulence effector. ROP18's specificity for Irga6 was experimentally demonstrated using phosphorylation assays against multiple IRG family members.","method":"Co-immunoprecipitation (GRA7–ROP5 binding), in vitro phosphorylation assay (panel of IRG proteins), IRG recruitment assay, genetic knockouts, mouse virulence assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro phosphorylation specificity panel plus interaction assay and in vivo validation; single lab, multiple methods","pmids":["26247512"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2015,"finding":"Deletion of ROP18 in South American type 4, 8, and 10 strains results in complete attenuation of virulence in mice (compared to only partial attenuation in type I RH), confirming that ROP18 is a conserved virulence factor across genetically divergent strains that evolved to resist innate immune defenses.","method":"CRISPR-Cas9 gene deletion, mouse virulence assay (multiple South American strains), genetic complementation","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO in multiple independent strain backgrounds with in vivo virulence readouts","pmids":["26291965"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"VAND (type 10); TgCtBr5 (type 8); TgCtBr18 (type 4)"}]},{"year":2009,"finding":"Three principal alleles of ROP18 exist across natural isolates; expression differences (driven by presence/absence of an upstream regulatory element) and allelic differences between these three alleles account for much of the variation in acute mouse virulence. Loss of the upstream element in the most ancient (type III) lineage eliminated ROP18 expression and virulence.","method":"Population genetics/sequencing of natural isolates, promoter deletion analysis, expression measurement, mouse virulence assay across isolates","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional promoter analysis combined with population data and virulence correlation; single lab","pmids":["19266027"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type II; type III; diverse natural isolates"}]},{"year":2011,"finding":"IFN-γ-dependent accumulation of murine Gbp1 on the PVM is regulated by the parasite virulence factors ROP16, ROP18, and GRA15. ROP18 (together with ROP5) prevents Gbp1 recruitment in a strain-dependent manner; all three factors were identified by systematic knockout comparison.","method":"Systematic knockout of ROP16, ROP18, GRA15; confocal imaging of mGBP1 localization; co-immunoprecipitation of mGBP2/4/5 with mGBP1","journal":"PLoS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, genetic knockouts with localization readout; ROP18 role confirmed by multiple parallel studies","pmids":["21931713"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type II; type III"}]},{"year":2013,"finding":"H. hammondi orthologs of TgROP5 and TgROP18 are functionally active when expressed in T. gondii: HhROP18 complemented a T. gondii background. A 107 bp promoter element absent in type III TgROP18 is necessary for ROP18 expression, indicating this element was active in the common ancestor of T. gondii and H. hammondi and was inactivated in the type III lineage progenitor.","method":"Genomic sequencing, cross-species complementation in T. gondii, promoter deletion analysis, mouse virulence assay","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cross-species complementation with promoter deletion; single lab","pmids":["23589877"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type III"},{"species":"Hammondia hammondi","strain":"HhCatGer041"}]},{"year":2014,"finding":"Expression of T. gondii ROP18 (TgROP18) in Neospora caninum (whose endogenous ROP18 is a pseudogene) significantly increases intracellular proliferation and virulence in mice, and the transgenic N. caninum can phosphorylate IRGs as T. gondii does, demonstrating that TgROP18 kinase activity is the key functional difference explaining virulence differences between these two related parasites.","method":"Transgenic expression of TgROP18 in N. caninum Nc1, mouse virulence assay, IRG phosphorylation assay, invasion/motility assays","journal":"PLoS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — heterologous gain-of-function complementation with in vitro and in vivo readouts; single lab","pmids":["24927100"],"is_preprint":false,"attribution":[{"species":"Neospora caninum","strain":"Nc1"}]},{"year":2018,"finding":"ROP18 phosphorylates host RTN1-C (an ER protein preferentially expressed in the CNS) at Ser7/134 and Thr4/8/118. ROP18-mediated phosphorylation of RTN1-C triggers ER stress-mediated apoptosis in neural cells and enhances GRP78 acetylation by attenuating HDAC activity, identifying a molecular mechanism for ROP18-dependent neuropathogenesis/encephalitis.","method":"Proteomics/mass spectrometry (active ROP18 pull-down from human proteome array), co-immunoprecipitation, in vitro phosphorylation (site-directed mapping), ER stress marker western blotting, HDAC activity assay","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics substrate identification plus co-IP and in vitro phosphorylation; single lab","pmids":["29784816"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2019,"finding":"ROP18 interacts with host cell P2X1 (an ATP-gated ion channel on neural cells) and inhibits ATP-induced apoptosis through the mitochondrial pathway by blocking P2X1-mediated Ca2+ influx, cytochrome C release, and caspase activation. This effect is cell-type specific (SF268 glioblastoma cells expressing P2X1, not RAW264.7 or THP-1 lacking P2X1).","method":"FRET, co-immunoprecipitation, flow cytometry (apoptosis/Ca2+ influx), western blotting (caspase/Bax/Bcl2/cytochrome C), overexpression experiments","journal":"Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple interaction and functional assays (FRET + Co-IP + functional readouts); single lab","pmids":["31164145"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2017,"finding":"ROP18 interacts with host cell vimentin and phosphorylates it. Vimentin knockout HBMEC cells show increased T. gondii infection rates, indicating that host vimentin restricts parasite invasion; ROP18-mediated phosphorylation alters vimentin solubility and dynamics. ROP18 contributes to regulation of vimentin phosphorylation and expression patterns during infection.","method":"Co-immunoprecipitation, in vitro kinase assay (vimentin phosphorylation), vimentin KO/knockdown cells, infection rate assay, western blotting","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP, in vitro phosphorylation, and KO cell assay; single lab","pmids":["29104504"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2017,"finding":"Using a human proteome array probed with bioactive recombinant ROP18, 68 putative host substrates were identified. Among validated targets, ROP18 phosphorylates Smad1 at Ser-187, triggering its proteasome-dependent degradation. ROP18 also targets p53, p38, and UBE2N for degradation.","method":"Human proteome array, in vitro kinase assay, phospho-site mutagenesis (Smad1 Ser-187), proteasome inhibitor experiments, western blotting for degradation","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — proteome-wide substrate screen validated by site-directed mutagenesis and degradation assay; single lab","pmids":["28087594"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2022,"finding":"ROP18 type I allele (TgROP18I) inhibits IFN-β production through direct interaction with IRF3, impeding the cGAS-STING signalling pathway. Additionally, TgROP18I interacts with p62 and TRAF6, inhibiting their interaction and p62 phosphorylation, thereby restricting ubiquitin/p62/LC3 recruitment to the PVM in both murine and human IFN-γ-stimulated cells.","method":"Co-immunoprecipitation, IFN-β reporter assay, western blotting (p62 phosphorylation, ubiquitin/LC3 on PVM), confocal microscopy","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple Co-IP interactions with functional signaling readouts; single lab","pmids":["35084749"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2021,"finding":"ROP18 type I allele (TgROP18I) interacts with human TRIM21 (an E3 ligase) and promotes its phosphorylation, causing TRIM21 degradation through the lysosomal pathway. Loss of TRIM21 reduces ubiquitin labeling on the PVM (preventing PV acidification and parasite killing), providing a mechanism for ROP18-dependent immune evasion in human cells.","method":"Co-immunoprecipitation, western blotting (TRIM21 degradation), lysosomal pathway inhibitor assay, TRIM21 knockdown/overexpression, confocal microscopy (PVM ubiquitination)","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with functional degradation and PVM ubiquitination assays; single lab","pmids":["34124071"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2016,"finding":"ROP18 association with the PVM is essential for ROP18-dependent antitumor immune activation (kinase-independent function), demonstrating a kinase-independent, PVM-localization-dependent role for ROP18 in host cell manipulation distinct from IRG evasion.","method":"Targeted deletion of ROP18, PVM-localization mutants, antitumor immunity assay in mouse ovarian cancer model","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PVM localization mutant distinguishes kinase-independent from kinase-dependent functions; single lab","pmids":["27447180"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type I uracil auxotroph (Δku80)"}]},{"year":2015,"finding":"ROP18 inhibits neural stem cell (C17.2 NSC) differentiation into neurons, at least partly by inhibiting the Wnt/β-catenin signalling pathway and downregulating Ngn1 and Ngn2. ROP18 reduces β-catenin levels and decreases Wnt/β-catenin transcriptional activity as measured by TOP/FOP flash reporter.","method":"Recombinant adenovirus (Ad-ROP18) transduction of C17.2 NSCs, immunoblot for βIII-tubulin/β-catenin, qRT-PCR for Ngn1/Ngn2, luciferase reporter (TOP/FOP flash)","journal":"Parasites & vectors","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, overexpression only without endogenous ROP18 manipulation or KO control; mechanism inferred from reporter assay","pmids":["29169404"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2025,"finding":"TgROP18 significantly reduces CD73 expression on decidual macrophages (dMφ) via the LSD1/SNAIL1 epigenetic pathway. Reduced CD73 subsequently decreases arginase-1 and IL-10 via the adenosine/A2AR/PKA/p-CREB/C/EBPβ pathway, impairing maternal-fetal immune tolerance. RH-Δrop18 infection caused less severe adverse pregnancy outcomes and less CD73 reduction than wild-type RH.","method":"RH-Δrop18 KO parasite infection of pregnant mice and human decidual tissues, flow cytometry (CD73/Arg-1/IL-10), western blot (LSD1/SNAIL1/signaling intermediates), ChIP assay","journal":"Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — KO parasite with multiple downstream mechanistic readouts and ChIP validation; single lab","pmids":["39994736"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2014,"finding":"NOTE — PMID:24648522 (Du et al., JBC, 'ROP18 inhibits NF-κB pathway by promoting p65 degradation') was subsequently WITHDRAWN by the journal and should not be used as evidence.","method":"WITHDRAWN paper — excluded","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Withdrawn paper — no valid evidence","pmids":["24648522"],"is_preprint":false,"attribution":[]},{"year":2020,"finding":"GRA60 localises to the vacuolar space and PVM and is found associated with ROP18 as part of the virulence complex. Deletion of GRA60 leads to IRG (Irgb10 and Irga6) recruitment to the PVM comparable to Δrop5, and double knockout GRA60/ROP5 parasites show IRG recruitment comparable to single knockouts, placing GRA60 in the same resistance pathway as ROP18.","method":"Confocal microscopy (IRG recruitment), co-immunoprecipitation (GRA60-ROP18 association), genetic knockout (ΔGra60, ΔGra60Δrop5), mouse virulence assay","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP interaction with KO epistasis and IRG recruitment assay; single lab","pmids":["33040458"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]}],"current_model":"ROP18 is a polymorphic serine-threonine kinase secreted from rhoptries into the host cell during T. gondii invasion, where it tethers to the cytosolic face of the parasitophorous vacuole membrane (PVM) via an arginine-rich N-terminal domain; at the PVM it operates within a multi-protein complex (ROP5 pseudokinase + ROP17 + GRA7 + GRA60) to phosphorylate and inactivate mouse immunity-related GTPases (specifically Irga6) on switch-I threonines, blocking IRG/GBP recruitment and PVM disruption; its N-terminal extension undergoes autophosphorylation to relieve autoinhibition, a second ligand-binding pocket outside the active site also contributes to virulence, and beyond IRG evasion, the kinase degrades host ATF6β (suppressing adaptive immunity), phosphorylates Smad1/p53/p38/vimentin/RTN1-C, and interacts with TRIM21, IRF3, and P2X1 to modulate host signalling, apoptosis, and immune responses in a strain-allele-dependent manner."},"narrative":{"mechanistic_narrative":"ROP18 is a polymorphic serine-threonine protein kinase secreted from Toxoplasma gondii rhoptries into the host cell during invasion, where it acts as a virulence effector that subverts host innate immunity and is essential for both acute pathogenesis and chronic infection [PMID:17170305, PMID:17170306, PMID:27165797]. Following secretion it relocalizes to the cytosolic face of the parasitophorous vacuole membrane (PVM), a tethering event mediated by its arginine-rich N-terminal domain; loss of this domain abolishes PVM association, IRG phosphorylation, and virulence despite normal secretion [PMID:17305424, PMID:22906355]. Its central function is the phosphorylation of mouse immunity-related GTPases (IRGs), specifically Irga6, on two conserved switch-I threonines, which biochemically inactivates the GTPase and prevents its accumulation on the PVM, thereby blocking clearance in IFN-γ-activated macrophages [PMID:21147463, PMID:21203588, PMID:26247512]. This activity operates within a multiprotein PVM complex: the ROP5 pseudokinase both regulates ROP18 catalytic activity and allosterically primes IRG substrates for phosphorylation, the co-active kinase ROP17 targets adjacent switch-I threonines, and dense granule proteins GRA7 and GRA60 act in the same IRG-resistance pathway [PMID:22802726, PMID:23144612, PMID:24390541, PMID:24832449, PMID:33040458]. ROP18 kinase activity is regulated by autophosphorylation of its N-terminal extension, which relieves autoinhibition, and a second ligand-binding pocket outside the active site independently contributes to virulence [PMID:19197235, PMID:24129568]. Beyond IRG evasion, ROP18 phosphorylates and destabilizes host targets including ATF6β to suppress CD8 T-cell responses, and phosphorylates Smad1, RTN1-C, and vimentin, linking it to modulation of host signalling, ER stress, and apoptosis [PMID:21670204, PMID:29784816, PMID:29104504, PMID:28087594]. Allelic and expression polymorphism—including loss of an upstream regulatory element in the type III lineage—accounts for much of the strain-dependent variation in virulence [PMID:19266027].","teleology":[{"year":2006,"claim":"Established that a single secreted parasite kinase can determine acute virulence, defining ROP18 as a genetically mapped effector rather than a passive antigen.","evidence":"QTL mapping and positional cloning with transfection of the virulent allele into a nonpathogenic type III strain and kinase-dead mutant analysis in a mouse virulence assay","pmids":["17170305","17170306"],"confidence":"High","gaps":["Host substrate(s) not yet identified","Mechanism connecting kinase activity to virulence unknown","Subcellular site of action not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I); type III"}]},{"year":2007,"claim":"Confirmed ROP18 is an intrinsically active serine-threonine kinase and localized it to rhoptries with re-targeting to the PVM upon invasion, placing the enzyme at the host-parasite interface.","evidence":"In vitro kinase assay with recombinant catalytic domain, transgenic overexpression with kinase-dead control, and immunofluorescence/fractionation in RH","pmids":["17305424"],"confidence":"High","gaps":["Identity of the ~70 kDa substrate not established","Mechanism of PVM re-localization unknown","No host substrate identified"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2009,"claim":"Defined how the kinase is switched on, showing autophosphorylation of the N-terminal extension relieves autoinhibition of the ATP-binding pocket.","evidence":"X-ray structures of ROP2/ROP8 pseudokinases used for homology modelling plus autophosphorylation assays, mutagenesis, and mass spectrometry","pmids":["19197235"],"confidence":"High","gaps":["Autoinhibition model later challenged by direct ROP18 kinase structure","Physiological ROP2/ROP8 phosphorylation significance unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Explained natural virulence variation by linking three ROP18 alleles and an upstream regulatory element to expression differences, showing loss of the element silenced ROP18 in the ancient type III lineage.","evidence":"Population sequencing of natural isolates, promoter deletion, expression measurement, and virulence assays across strains","pmids":["19266027"],"confidence":"Medium","gaps":["Molecular basis of allelic activity differences not fully resolved","Regulatory element trans-factors unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type II; type III; diverse natural isolates"}]},{"year":2010,"claim":"Identified the key host substrate class, showing ROP18 phosphorylates IRGs (Irga6) on switch-I threonines to inactivate them and block PVM recruitment, defining the mechanism of macrophage evasion.","evidence":"Co-IP, in vitro phosphorylation with substrate profiling, Δrop18 knockout, IFN-γ macrophage clearance and mouse virulence assays; phospho-site mapping and GTPase activity readouts","pmids":["21147463","21203588"],"confidence":"High","gaps":["Full IRG substrate range not yet delimited","Requirement for co-factors not yet appreciated","Human-cell relevance unaddressed"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"},{"species":"Toxoplasma gondii","strain":"type I; type II; type III"}]},{"year":2011,"claim":"Extended ROP18's targets beyond IRGs to the host ER transcription factor ATF6β, linking the kinase to suppression of adaptive (CD8 T-cell) immunity.","evidence":"Reciprocal Co-IP, mammalian overexpression, Δrop18, proteasome inhibition, and ATF6β-KO mouse challenge in RH","pmids":["21670204"],"confidence":"High","gaps":["Direct phospho-site on ATF6β not mapped","E3 ligase mediating degradation unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2011,"claim":"Placed ROP18 genetically upstream of GBP-mediated killing and revealed its dependence on ROP5, showing both are required to prevent Gbp1 recruitment.","evidence":"Δrop18/Δrop5 knockouts, Gbp1−/− macrophages and mice, systematic ROP16/ROP18/GRA15 knockout comparison with confocal imaging","pmids":["23633952","21931713"],"confidence":"High","gaps":["Direct versus indirect effect on GBPs vs IRGs not separated","Mechanism of ROP5 dependence not yet defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"},{"species":"Toxoplasma gondii","strain":"type I; type II; type III"}]},{"year":2012,"claim":"Resolved the co-factor requirement, showing ROP5 pseudokinases directly regulate ROP18 catalytic activity and allosterically prime IRG substrates, and that PVM tethering via the arginine-rich N-terminus is essential for function.","evidence":"Allelic swaps, structural/biochemical IRG binding and oligomerization assays, in vitro kinase activity assays, N-terminal deletion mutagenesis with virulence readouts, and human vs mouse cell comparison","pmids":["22761577","22802726","23144612","22906355"],"confidence":"High","gaps":["Structural detail of the ROP5–ROP18–IRG ternary arrangement incomplete","Human-cell IRG-independent function undefined at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type I; type II; type III"},{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2013,"claim":"Refined the activation model by determining the ROP18 kinase-domain crystal structure, which contradicted the earlier autoinhibition model and revealed a second ligand-binding pocket required for virulence.","evidence":"X-ray crystallography with sucrose-bound pocket, mutagenesis of the second pocket, and mouse virulence assays in RH","pmids":["24129568"],"confidence":"High","gaps":["Physiological ligand of the second pocket unknown","Reconciliation with N-terminal autophosphorylation model incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2013,"claim":"Demonstrated cross-species conservation and regulatory-element origin, showing H. hammondi ROP18 complements T. gondii and that the promoter element silenced in type III was ancestrally active.","evidence":"Genomic sequencing, cross-species complementation in T. gondii, promoter deletion, and virulence assays","pmids":["23589877"],"confidence":"Medium","gaps":["Functional divergence of H. hammondi complex components unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type III"},{"species":"Hammondia hammondi","strain":"HhCatGer041"}]},{"year":2014,"claim":"Defined the full PVM virulence complex, showing ROP18 associates with ROP5, ROP17, ROP2/ROP8, and GRA7, and that ROP17 and ROP18 cooperatively phosphorylate adjacent switch-I threonines.","evidence":"Tandem affinity purification-MS in T. gondii, Co-IP, in vitro kinase assays, single and double knockouts, IRG polymerization and recruitment assays in RH","pmids":["24390541","24832449"],"confidence":"High","gaps":["Stoichiometry and assembly order of the complex not resolved","Division of labor among kinases only partially defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2014,"claim":"Showed via heterologous gain-of-function that ROP18 kinase activity is the decisive functional difference distinguishing virulent T. gondii from related parasites carrying a ROP18 pseudogene.","evidence":"Transgenic TgROP18 expression in Neospora caninum with mouse virulence and IRG phosphorylation assays","pmids":["24927100"],"confidence":"Medium","gaps":["Whether N. caninum provides all cognate co-factors unclear"],"attribution":[{"species":"Neospora caninum","strain":"Nc1"}]},{"year":2015,"claim":"Established Irga6 as the specific ROP18 target and clarified GRA7's role, showing GRA7 tethers to the PVM via ROP5 and enhances Irga6 phosphorylation.","evidence":"Co-IP (GRA7–ROP5), in vitro phosphorylation against a panel of IRG proteins, knockouts, and mouse virulence assays in RH","pmids":["26247512"],"confidence":"High","gaps":["Why other IRGs are not phosphorylated not fully explained mechanistically"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2015,"claim":"Broadened the evolutionary significance of ROP18 by showing complete virulence attenuation upon deletion across divergent South American strains, contrasting with partial attenuation in RH.","evidence":"CRISPR-Cas9 deletion and complementation in type 4, 8, and 10 strains with mouse virulence assays","pmids":["26291965"],"confidence":"High","gaps":["Strain-specific co-factor differences underlying the phenotype not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"VAND (type 10); TgCtBr5 (type 8); TgCtBr18 (type 4)"}]},{"year":2016,"claim":"Uncovered a kinase-independent, PVM-localization-dependent role for ROP18 in host manipulation, distinct from its IRG-phosphorylating function.","evidence":"Δrop18 and PVM-localization mutants in an antitumor immunity mouse ovarian cancer model","pmids":["27447180"],"confidence":"Medium","gaps":["Molecular effector of the kinase-independent function unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I uracil auxotroph (Δku80)"}]},{"year":2016,"claim":"Extended ROP18's essentiality to chronic infection, showing kinase activity and PVM association are required for IRG resistance and cyst formation in type II.","evidence":"Systematic ROPK-locus deletions, kinase-dead and localization mutants, and cyst-burden/virulence assays in ME49 (type II)","pmids":["27165797"],"confidence":"High","gaps":["Type II vs type I mechanistic differences in cyst context not fully resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49 (type II)"}]},{"year":2017,"claim":"Systematically expanded the host substrate repertoire via proteome-array screening, identifying Smad1 (Ser-187), p53, p38, and UBE2N and vimentin as ROP18 targets linked to degradation and invasion control.","evidence":"Human proteome array probed with recombinant ROP18, in vitro kinase assays, phospho-site mutagenesis, proteasome inhibition, and vimentin-KO infection assays in RH","pmids":["28087594","29104504"],"confidence":"Medium","gaps":["Which substrates are phosphorylated in vivo during infection not established","Physiological consequences of most targets not fully mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2018,"claim":"Connected ROP18 to CNS pathology by identifying RTN1-C as a phospho-substrate whose modification triggers ER-stress apoptosis in neural cells.","evidence":"Proteome-array pull-down, Co-IP, in vitro phosphorylation with site mapping, ER-stress markers, and HDAC activity assays in RH","pmids":["29784816"],"confidence":"Medium","gaps":["In vivo relevance to encephalitis not directly established","Link between phosphorylation and GRP78 acetylation mechanistically incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2019,"claim":"Revealed a cell-type-specific anti-apoptotic function, showing ROP18 interacts with the ATP-gated channel P2X1 to block mitochondrial apoptosis.","evidence":"FRET, Co-IP, flow cytometry for apoptosis/Ca2+ influx, and apoptosis-marker western blotting in P2X1-expressing SF268 cells in RH","pmids":["31164145"],"confidence":"Medium","gaps":["Whether interaction requires kinase activity unclear","In vivo relevance not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2020,"claim":"Added GRA60 to the IRG-resistance pathway, showing it associates with ROP18 and its loss phenocopies Δrop5 for IRG recruitment.","evidence":"Co-IP, single and double knockouts, IRG (Irgb10/Irga6) recruitment confocal assays, and virulence assays in RH","pmids":["33040458"],"confidence":"Medium","gaps":["Direct biochemical role of GRA60 in the complex undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2021,"claim":"Defined ROP18-mediated evasion in human cells by targeting TRIM21 for lysosomal degradation, reducing PVM ubiquitination and parasite killing.","evidence":"Co-IP, degradation and lysosomal-inhibitor assays, TRIM21 knockdown/overexpression, and PVM ubiquitination confocal microscopy for TgROP18I in RH","pmids":["34124071"],"confidence":"Medium","gaps":["Direct phosphorylation of TRIM21 not conclusively mapped","Relationship to IRG-independent human resistance incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2022,"claim":"Showed ROP18 suppresses type I interferon and autophagic PVM targeting by interacting with IRF3, p62, and TRAF6 in both murine and human cells.","evidence":"Co-IP, IFN-β reporter assay, p62 phosphorylation and PVM ubiquitin/LC3 western blotting and confocal microscopy for TgROP18I","pmids":["35084749"],"confidence":"Medium","gaps":["Whether these interactions require kinase activity not resolved","In vivo contribution not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":2025,"claim":"Linked ROP18 to reproductive pathology, showing it lowers CD73 on decidual macrophages via LSD1/SNAIL1 signalling to impair maternal-fetal immune tolerance.","evidence":"RH-Δrop18 infection of pregnant mice and human decidual tissue, flow cytometry, western blotting, and ChIP assays in RH","pmids":["39994736"],"confidence":"Medium","gaps":["Whether the effect is direct or downstream of general infection burden not fully separated","Kinase-dependence not established"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]},{"year":null,"claim":"How ROP18's many reported host substrates and interactions (Smad1, p53, p38, RTN1-C, vimentin, TRIM21, IRF3, P2X1) are prioritized and coordinated in vivo, and how kinase-dependent versus kinase-independent PVM functions are integrated, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model separating in-vivo-relevant substrates from proteome-array hits","Structural basis reconciling autophosphorylation activation with the crystal-structure autoinhibition rebuttal not settled","Kinase-independent PVM effector remains unidentified"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["ROP18–ROP5 pseudokinase–ROP17–GRA7 PVM virulence complex","ROP18–ROP2/ROP8–GRA7 PVM complex","ROP18–GRA60 IRG-resistance association"],"partners":["ROP5 (PSEUDOKINASE CO-FACTOR AND CATALYTIC REGULATOR)","ROP17 (CO-ACTIVE KINASE, ADJACENT SWITCH-I THREONINES)","GRA7 (DENSE GRANULE PROTEIN, IRGA6 BINDING)","GRA60 (DENSE GRANULE PROTEIN, IRG-RESISTANCE PATHWAY)","IRGA6 / IMMUNITY-RELATED GTPASES (SUBSTRATE)","ATF6Β (HOST, DEGRADED)","SMAD1 / P53 / P38 / UBE2N (HOST SUBSTRATES)","RTN1-C (HOST SUBSTRATE)","VIMENTIN (HOST SUBSTRATE)","TRIM21 (HOST, DEGRADED)","IRF3, P62, TRAF6 (HOST SIGNALLING)","P2X1 (HOST ION CHANNEL)"],"other_free_text":["Activation involves autophosphorylation of the N-terminal extension relieving autoinhibition (#2); a later crystal structure disputes this autoinhibition model and defines a second ligand-binding pocket required for virulence (#11).","PVM tethering is mediated by an arginine-rich N-terminal domain and is essential for both kinase-dependent IRG evasion and a kinase-independent host-manipulation function (#7, #27).","Polymorphism: three principal alleles and an upstream regulatory element govern expression and virulence; type III lost the element and ROP18 expression (#17, #19).","PMID:24648522 (NF-κB/p65 degradation) was withdrawn and is excluded from evidence (#30)."]},"strain_conflicts":[{"aspect":"Virulence dependence on ROP18 (attenuation upon deletion)","claims":[{"attribution":[{"species":"Toxoplasma gondii","strain":"RH (type I)"}],"claim":"Δrop18 causes only partial attenuation of acute virulence in type I RH","supporting_discovery_ids":[16]},{"attribution":[{"species":"Toxoplasma gondii","strain":"VAND (type 10); TgCtBr5 (type 8); TgCtBr18 (type 4)"}],"claim":"Deletion of ROP18 causes complete attenuation of virulence in South American type 4, 8, and 10 strains","supporting_discovery_ids":[16]},{"attribution":[{"species":"Toxoplasma gondii","strain":"ME49 (type II)"}],"claim":"ROP18 kinase activity and PVM association are required for IRG resistance and chronic cyst formation in type II","supporting_discovery_ids":[14]}]},{"aspect":"ROP18 expression across natural alleles","claims":[{"attribution":[{"species":"Toxoplasma gondii","strain":"type I; type II"}],"claim":"ROP18 is expressed and confers virulence, with allele-dependent activity","supporting_discovery_ids":[17,4]},{"attribution":[{"species":"Toxoplasma gondii","strain":"type III"}],"claim":"Loss of an upstream regulatory element eliminated ROP18 expression and virulence","supporting_discovery_ids":[17,19]}]},{"aspect":"Irga6 phosphorylation efficiency by ROP18 allele","claims":[{"attribution":[{"species":"Toxoplasma gondii","strain":"type I"}],"claim":"The virulent type I allele efficiently phosphorylates mouse Irga6 at two switch-I threonines","supporting_discovery_ids":[4]},{"attribution":[{"species":"Toxoplasma gondii","strain":"type II; type III"}],"claim":"Phosphorylation of Irga6 is markedly weaker (type I > type II/III), correlating with lower virulence","supporting_discovery_ids":[4]}]}]},"prefetch_data":{},"corpus_meta":[{"pmid":"17170306","id":"PMC_17170306","title":"Polymorphic secreted kinases are key virulence factors in toxoplasmosis.","date":"2006","source":"Science (New York, 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serine-threonine kinase secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4–5 logs, and these attributes required kinase activity, establishing ROP18 as a secreted effector kinase essential for acute virulence.\",\n      \"method\": \"Genetic mapping (QTL), positional cloning, transfection/complementation in type III strain, kinase-dead mutant analysis, mouse virulence assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — complementation in vivo with kinase-dead mutagenesis, replicated across two independent papers (PMID:17170305, PMID:17170306)\",\n      \"pmids\": [\"17170305\", \"17170306\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I); type III\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Recombinant ROP18 catalytic domain (aa 243–539) possesses serine-threonine protein kinase activity in vitro and phosphorylates a ~70 kDa tachyzoite substrate. Overexpression of ROP18 in transgenic parasites dramatically increases intra-vacuolar parasite multiplication rate in a kinase-activity-dependent manner. ROP18 localises to rhoptries and re-localises to the parasitophorous vacuole membrane (PVM) upon invasion.\",\n      \"method\": \"In vitro kinase assay with recombinant protein, transgenic overexpression, kinase-dead mutant, immunofluorescence/subcellular fractionation\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with recombinant catalytic domain plus mutagenesis and live-parasite phenotype in a single rigorous study\",\n      \"pmids\": [\"17305424\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"X-ray crystal structures of pseudokinases ROP2 and ROP8 provided a template for homology modelling of ROP18. Autophosphorylation of key residues in the N-terminal extension activates ROP18; this phosphorylated N-terminus relieves autoinhibition caused by aliphatic side-chain insertion into the ATP-binding pocket. Mutagenesis and mass spectrometry confirmed this activation mechanism, and activated ROP18 in turn phosphorylates ROP2 and ROP8.\",\n      \"method\": \"X-ray crystallography, homology modelling, autophosphorylation assay, mutagenesis, mass spectrometry\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis and mass spectrometry in one rigorous study\",\n      \"pmids\": [\"19197235\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ROP18 binds to and phosphorylates multiple immunity-related GTPases (IRGs) on a conserved motif within switch region I of the GTPase domain. This modification prevents IRG recruitment to the PVM, blocks clearance within IFN-γ-activated macrophages and inflammatory monocytes, and is necessary and sufficient to confer parasite survival and virulence in vivo. Substrate profiling revealed the ROP18 phosphorylation consensus.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphorylation assay, substrate profiling, genetic knockout (Δrop18), IFN-γ-activated macrophage clearance assay, mouse virulence assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with substrate profiling, reciprocal interaction, KO phenotype, in vivo validation; replicated in same year by PMID:21203588\",\n      \"pmids\": [\"21147463\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The type I virulent allele of ROP18 phosphorylates mouse Irga6 at two threonines in the nucleotide-binding domain (switch I region), biochemically inactivating the GTPase and inhibiting its accumulation at the PVM. This phosphorylation event is strain-specific (type I > type II/III) and correlates with virulence in mice.\",\n      \"method\": \"In vitro phosphorylation assay, phospho-site mapping, GTPase activity assay, confocal microscopy of IRG recruitment, mouse virulence comparison\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical phosphorylation with site mapping and functional GTPase readout, independent replication of IRG targeting alongside PMID:21147463\",\n      \"pmids\": [\"21203588\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ROP18 targets the host ER-bound transcription factor ATF6β. The N-terminal extension of ROP18 interacts with ATF6β and destabilizes it; ROP18 kinase activity is required for proteasome-dependent degradation of ATF6β, which downregulates CD8 T cell-mediated adaptive immune responses. ATF6β-deficient mice are highly susceptible to ROP18-deficient parasites, confirming this axis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, overexpression in mammalian cells, ROP18 gene disruption (Δrop18), proteasome inhibitor experiments, ATF6β-KO mice challenge\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, KO parasite phenotype, KO host mouse validation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"21670204\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ROP18 (active kinase) and ROP5 (pseudokinase) are both required for avoidance of Gbp1 recruitment to the PVM; virulent strains lacking either ROP18 or ROP5 show increased Gbp1 accumulation and clearance in IFN-γ-activated macrophages. The virulence defect of Δrop18 mutants was reversed in Gbp1−/− cells and mice, demonstrating that ROP18 function is upstream of Gbp1-mediated killing.\",\n      \"method\": \"Genetic knockouts (Δrop18, Δrop5), IFN-γ-activated macrophage clearance assay, Gbp1−/− mice, confocal microscopy\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO parasites, KO host cells, in vivo KO mice; replicated across labs\",\n      \"pmids\": [\"23633952\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The arginine-rich N-terminal domain of ROP18 (upstream of the kinase domain) is required for tethering ROP18 to the cytosolic face of the PVM following secretion. Mutants lacking this domain are secreted but fail to associate with the PVM, cannot phosphorylate Irga6, cannot prevent IRG loading onto the PVM, and are avirulent in mice, demonstrating that PVM targeting is essential for ROP18 function.\",\n      \"method\": \"N-terminal deletion/mutagenesis, confocal microscopy, in vitro phosphorylation assay (Irga6), mouse virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — domain mutagenesis linked to localization, substrate phosphorylation, and in vivo virulence in a single focused study\",\n      \"pmids\": [\"22906355\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROP18 can only efficiently inhibit IRG accumulation on the PVM in strains that co-express virulent ROP5 alleles. ROP5 can directly interact with one or more IRGs and can reduce IRG coating even in the absence of ROP18. Neither ROP18 nor ROP5 markedly affect survival in IFN-γ-activated human cells (which lack the IRG system), indicating that ROP18/ROP5 evolved specifically to block murine IRGs.\",\n      \"method\": \"Genetic knockouts and allelic swaps, IRG recruitment assay, human cell infection assay, mouse virulence assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple allelic combinations tested across strains, species comparison (mouse vs. human cells), replicated by multiple labs\",\n      \"pmids\": [\"22761577\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROP5 proteins (pseudokinases) act as essential co-factors for ROP18 by binding adjacent to the nucleotide-binding domain of IRG targets and inducing an allosteric change: this prevents IRG oligomerization and reorients the two threonine residues in switch I that are targeted for phosphorylation by ROP18, thereby preparing the substrate for phosphorylation by ROP18.\",\n      \"method\": \"Genetic and biochemical binding assays, structural analysis (ROP5 pseudokinase domain), IRG oligomerization assays, in vitro phosphorylation\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — structural + biochemical + genetic epistasis, replicated across multiple papers (PMID:22802726, PMID:23144612, PMID:25118287)\",\n      \"pmids\": [\"22802726\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROP5 directly regulates the catalytic activity of ROP18 in vitro. Both ROP5 and ROP18 are necessary to avoid IRG recruitment and clearance in IFN-γ-activated macrophages. The virulence defects of Δrop5 and Δrop18 were fully restored in Irgm3−/− mice, placing ROP18 activity genetically downstream of ROP5 and upstream of Irgm3-dependent IRG function.\",\n      \"method\": \"In vitro kinase activity assay (ROP18 ± ROP5), genetic knockouts, macrophage clearance assay, Irgm3−/− mice epistasis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro activity regulation plus genetic epistasis in KO hosts; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"23144612\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of the ROP18 kinase domain was determined. The structure is inconsistent with a previously proposed autoinhibitory mechanism. A sucrose molecule bound outside the active-site cleft identifies a second ligand-binding pocket; mutational analysis confirmed that this pocket is important for acute virulence in mice.\",\n      \"method\": \"X-ray crystallography, mutagenesis of second ligand-binding pocket, mouse virulence assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis with in vivo functional validation in a single study\",\n      \"pmids\": [\"24129568\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROP18 exists in a protein complex with pseudokinases ROP8/ROP2 and the dense granule protein GRA7 at the PVM. GRA7 directly binds the active dimer of Irga6 in a GTP-dependent manner, leading to enhanced IRG polymerization and rapid turnover/disassembly. ROP18 and GRA7 act together (synergistically and in the same pathway) to target IRGs by distinct mechanisms; a combined gra7/rop18 double knockout is avirulent while single mutants are only partially attenuated.\",\n      \"method\": \"Tandem affinity purification-MS, co-immunoprecipitation, in vitro GTPase/polymerization assay, genetic knockouts (Δgra7, Δrop18, double KO), IRG recruitment assay, mouse virulence assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS complex identification confirmed by Co-IP, biochemical IRG assay, and double-KO epistasis; single lab\",\n      \"pmids\": [\"24390541\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROP5 forms physical complexes with the active kinases ROP18 and ROP17 (identified by tandem affinity purification in T. gondii). ROP18 and ROP17 together target adjacent essential threonine residues in switch I of IRGs; combined activities of ROP17 and ROP18 are required to prevent IRG recruitment to the PVM and protect the parasite in IFN-γ-activated macrophages.\",\n      \"method\": \"Tandem affinity purification-MS in T. gondii, in vitro kinase assay, genetic knockouts, IRG recruitment/macrophage clearance assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in-parasite complex purification with MS plus biochemical and genetic validation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"24832449\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROP18 kinase activity and PVM association are both necessary for resistance to host IRGs in type II T. gondii. Deletion of ROP18 (and ROP5, ROP17) in type II severely reduces cyst burdens and >100-fold reduces virulence, establishing ROP18 as essential for chronic infection and not only acute pathogenesis.\",\n      \"method\": \"Targeted gene deletion of 26 ROPK loci, kinase-dead mutant, PVM-localization mutant, IRG resistance assay, mouse virulence/cyst burden assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic KO with kinase-dead and localization mutants, in vivo chronic infection readout; single lab but comprehensive\",\n      \"pmids\": [\"27165797\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49 (type II)\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA7 association with the PVM is mediated by direct binding to ROP5, and GRA7 absence results in reduced phosphorylation of Irga6, increased IRG amounts on the vacuole, and attenuated virulence. Among all IRG proteins, Irga6 is the only specific target of ROP18; similarly, GRA7 is strictly an Irga6-specific virulence effector. ROP18's specificity for Irga6 was experimentally demonstrated using phosphorylation assays against multiple IRG family members.\",\n      \"method\": \"Co-immunoprecipitation (GRA7–ROP5 binding), in vitro phosphorylation assay (panel of IRG proteins), IRG recruitment assay, genetic knockouts, mouse virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phosphorylation specificity panel plus interaction assay and in vivo validation; single lab, multiple methods\",\n      \"pmids\": [\"26247512\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Deletion of ROP18 in South American type 4, 8, and 10 strains results in complete attenuation of virulence in mice (compared to only partial attenuation in type I RH), confirming that ROP18 is a conserved virulence factor across genetically divergent strains that evolved to resist innate immune defenses.\",\n      \"method\": \"CRISPR-Cas9 gene deletion, mouse virulence assay (multiple South American strains), genetic complementation\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO in multiple independent strain backgrounds with in vivo virulence readouts\",\n      \"pmids\": [\"26291965\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"VAND (type 10); TgCtBr5 (type 8); TgCtBr18 (type 4)\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Three principal alleles of ROP18 exist across natural isolates; expression differences (driven by presence/absence of an upstream regulatory element) and allelic differences between these three alleles account for much of the variation in acute mouse virulence. Loss of the upstream element in the most ancient (type III) lineage eliminated ROP18 expression and virulence.\",\n      \"method\": \"Population genetics/sequencing of natural isolates, promoter deletion analysis, expression measurement, mouse virulence assay across isolates\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional promoter analysis combined with population data and virulence correlation; single lab\",\n      \"pmids\": [\"19266027\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III; diverse natural isolates\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IFN-γ-dependent accumulation of murine Gbp1 on the PVM is regulated by the parasite virulence factors ROP16, ROP18, and GRA15. ROP18 (together with ROP5) prevents Gbp1 recruitment in a strain-dependent manner; all three factors were identified by systematic knockout comparison.\",\n      \"method\": \"Systematic knockout of ROP16, ROP18, GRA15; confocal imaging of mGBP1 localization; co-immunoprecipitation of mGBP2/4/5 with mGBP1\",\n      \"journal\": \"PLoS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, genetic knockouts with localization readout; ROP18 role confirmed by multiple parallel studies\",\n      \"pmids\": [\"21931713\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"H. hammondi orthologs of TgROP5 and TgROP18 are functionally active when expressed in T. gondii: HhROP18 complemented a T. gondii background. A 107 bp promoter element absent in type III TgROP18 is necessary for ROP18 expression, indicating this element was active in the common ancestor of T. gondii and H. hammondi and was inactivated in the type III lineage progenitor.\",\n      \"method\": \"Genomic sequencing, cross-species complementation in T. gondii, promoter deletion analysis, mouse virulence assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cross-species complementation with promoter deletion; single lab\",\n      \"pmids\": [\"23589877\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type III\"}, {\"species\": \"Hammondia hammondi\", \"strain\": \"HhCatGer041\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Expression of T. gondii ROP18 (TgROP18) in Neospora caninum (whose endogenous ROP18 is a pseudogene) significantly increases intracellular proliferation and virulence in mice, and the transgenic N. caninum can phosphorylate IRGs as T. gondii does, demonstrating that TgROP18 kinase activity is the key functional difference explaining virulence differences between these two related parasites.\",\n      \"method\": \"Transgenic expression of TgROP18 in N. caninum Nc1, mouse virulence assay, IRG phosphorylation assay, invasion/motility assays\",\n      \"journal\": \"PLoS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — heterologous gain-of-function complementation with in vitro and in vivo readouts; single lab\",\n      \"pmids\": [\"24927100\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Neospora caninum\", \"strain\": \"Nc1\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ROP18 phosphorylates host RTN1-C (an ER protein preferentially expressed in the CNS) at Ser7/134 and Thr4/8/118. ROP18-mediated phosphorylation of RTN1-C triggers ER stress-mediated apoptosis in neural cells and enhances GRP78 acetylation by attenuating HDAC activity, identifying a molecular mechanism for ROP18-dependent neuropathogenesis/encephalitis.\",\n      \"method\": \"Proteomics/mass spectrometry (active ROP18 pull-down from human proteome array), co-immunoprecipitation, in vitro phosphorylation (site-directed mapping), ER stress marker western blotting, HDAC activity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics substrate identification plus co-IP and in vitro phosphorylation; single lab\",\n      \"pmids\": [\"29784816\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ROP18 interacts with host cell P2X1 (an ATP-gated ion channel on neural cells) and inhibits ATP-induced apoptosis through the mitochondrial pathway by blocking P2X1-mediated Ca2+ influx, cytochrome C release, and caspase activation. This effect is cell-type specific (SF268 glioblastoma cells expressing P2X1, not RAW264.7 or THP-1 lacking P2X1).\",\n      \"method\": \"FRET, co-immunoprecipitation, flow cytometry (apoptosis/Ca2+ influx), western blotting (caspase/Bax/Bcl2/cytochrome C), overexpression experiments\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple interaction and functional assays (FRET + Co-IP + functional readouts); single lab\",\n      \"pmids\": [\"31164145\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ROP18 interacts with host cell vimentin and phosphorylates it. Vimentin knockout HBMEC cells show increased T. gondii infection rates, indicating that host vimentin restricts parasite invasion; ROP18-mediated phosphorylation alters vimentin solubility and dynamics. ROP18 contributes to regulation of vimentin phosphorylation and expression patterns during infection.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay (vimentin phosphorylation), vimentin KO/knockdown cells, infection rate assay, western blotting\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP, in vitro phosphorylation, and KO cell assay; single lab\",\n      \"pmids\": [\"29104504\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Using a human proteome array probed with bioactive recombinant ROP18, 68 putative host substrates were identified. Among validated targets, ROP18 phosphorylates Smad1 at Ser-187, triggering its proteasome-dependent degradation. ROP18 also targets p53, p38, and UBE2N for degradation.\",\n      \"method\": \"Human proteome array, in vitro kinase assay, phospho-site mutagenesis (Smad1 Ser-187), proteasome inhibitor experiments, western blotting for degradation\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — proteome-wide substrate screen validated by site-directed mutagenesis and degradation assay; single lab\",\n      \"pmids\": [\"28087594\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ROP18 type I allele (TgROP18I) inhibits IFN-β production through direct interaction with IRF3, impeding the cGAS-STING signalling pathway. Additionally, TgROP18I interacts with p62 and TRAF6, inhibiting their interaction and p62 phosphorylation, thereby restricting ubiquitin/p62/LC3 recruitment to the PVM in both murine and human IFN-γ-stimulated cells.\",\n      \"method\": \"Co-immunoprecipitation, IFN-β reporter assay, western blotting (p62 phosphorylation, ubiquitin/LC3 on PVM), confocal microscopy\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple Co-IP interactions with functional signaling readouts; single lab\",\n      \"pmids\": [\"35084749\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ROP18 type I allele (TgROP18I) interacts with human TRIM21 (an E3 ligase) and promotes its phosphorylation, causing TRIM21 degradation through the lysosomal pathway. Loss of TRIM21 reduces ubiquitin labeling on the PVM (preventing PV acidification and parasite killing), providing a mechanism for ROP18-dependent immune evasion in human cells.\",\n      \"method\": \"Co-immunoprecipitation, western blotting (TRIM21 degradation), lysosomal pathway inhibitor assay, TRIM21 knockdown/overexpression, confocal microscopy (PVM ubiquitination)\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with functional degradation and PVM ubiquitination assays; single lab\",\n      \"pmids\": [\"34124071\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ROP18 association with the PVM is essential for ROP18-dependent antitumor immune activation (kinase-independent function), demonstrating a kinase-independent, PVM-localization-dependent role for ROP18 in host cell manipulation distinct from IRG evasion.\",\n      \"method\": \"Targeted deletion of ROP18, PVM-localization mutants, antitumor immunity assay in mouse ovarian cancer model\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PVM localization mutant distinguishes kinase-independent from kinase-dependent functions; single lab\",\n      \"pmids\": [\"27447180\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I uracil auxotroph (Δku80)\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ROP18 inhibits neural stem cell (C17.2 NSC) differentiation into neurons, at least partly by inhibiting the Wnt/β-catenin signalling pathway and downregulating Ngn1 and Ngn2. ROP18 reduces β-catenin levels and decreases Wnt/β-catenin transcriptional activity as measured by TOP/FOP flash reporter.\",\n      \"method\": \"Recombinant adenovirus (Ad-ROP18) transduction of C17.2 NSCs, immunoblot for βIII-tubulin/β-catenin, qRT-PCR for Ngn1/Ngn2, luciferase reporter (TOP/FOP flash)\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, overexpression only without endogenous ROP18 manipulation or KO control; mechanism inferred from reporter assay\",\n      \"pmids\": [\"29169404\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TgROP18 significantly reduces CD73 expression on decidual macrophages (dMφ) via the LSD1/SNAIL1 epigenetic pathway. Reduced CD73 subsequently decreases arginase-1 and IL-10 via the adenosine/A2AR/PKA/p-CREB/C/EBPβ pathway, impairing maternal-fetal immune tolerance. RH-Δrop18 infection caused less severe adverse pregnancy outcomes and less CD73 reduction than wild-type RH.\",\n      \"method\": \"RH-Δrop18 KO parasite infection of pregnant mice and human decidual tissues, flow cytometry (CD73/Arg-1/IL-10), western blot (LSD1/SNAIL1/signaling intermediates), ChIP assay\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — KO parasite with multiple downstream mechanistic readouts and ChIP validation; single lab\",\n      \"pmids\": [\"39994736\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NOTE — PMID:24648522 (Du et al., JBC, 'ROP18 inhibits NF-κB pathway by promoting p65 degradation') was subsequently WITHDRAWN by the journal and should not be used as evidence.\",\n      \"method\": \"WITHDRAWN paper — excluded\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Withdrawn paper — no valid evidence\",\n      \"pmids\": [\"24648522\"],\n      \"is_preprint\": false,\n      \"attribution\": []\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA60 localises to the vacuolar space and PVM and is found associated with ROP18 as part of the virulence complex. Deletion of GRA60 leads to IRG (Irgb10 and Irga6) recruitment to the PVM comparable to Δrop5, and double knockout GRA60/ROP5 parasites show IRG recruitment comparable to single knockouts, placing GRA60 in the same resistance pathway as ROP18.\",\n      \"method\": \"Confocal microscopy (IRG recruitment), co-immunoprecipitation (GRA60-ROP18 association), genetic knockout (ΔGra60, ΔGra60Δrop5), mouse virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP interaction with KO epistasis and IRG recruitment assay; single lab\",\n      \"pmids\": [\"33040458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    }\n  ],\n  \"current_model\": \"ROP18 is a polymorphic serine-threonine kinase secreted from rhoptries into the host cell during T. gondii invasion, where it tethers to the cytosolic face of the parasitophorous vacuole membrane (PVM) via an arginine-rich N-terminal domain; at the PVM it operates within a multi-protein complex (ROP5 pseudokinase + ROP17 + GRA7 + GRA60) to phosphorylate and inactivate mouse immunity-related GTPases (specifically Irga6) on switch-I threonines, blocking IRG/GBP recruitment and PVM disruption; its N-terminal extension undergoes autophosphorylation to relieve autoinhibition, a second ligand-binding pocket outside the active site also contributes to virulence, and beyond IRG evasion, the kinase degrades host ATF6β (suppressing adaptive immunity), phosphorylates Smad1/p53/p38/vimentin/RTN1-C, and interacts with TRIM21, IRF3, and P2X1 to modulate host signalling, apoptosis, and immune responses in a strain-allele-dependent manner.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ROP18 is a polymorphic serine-threonine protein kinase secreted from Toxoplasma gondii rhoptries into the host cell during invasion, where it acts as a virulence effector that subverts host innate immunity and is essential for both acute pathogenesis and chronic infection [#0, #14]. Following secretion it relocalizes to the cytosolic face of the parasitophorous vacuole membrane (PVM), a tethering event mediated by its arginine-rich N-terminal domain; loss of this domain abolishes PVM association, IRG phosphorylation, and virulence despite normal secretion [#1, #7]. Its central function is the phosphorylation of mouse immunity-related GTPases (IRGs), specifically Irga6, on two conserved switch-I threonines, which biochemically inactivates the GTPase and prevents its accumulation on the PVM, thereby blocking clearance in IFN-\\u03b3-activated macrophages [#3, #4, #15]. This activity operates within a multiprotein PVM complex: the ROP5 pseudokinase both regulates ROP18 catalytic activity and allosterically primes IRG substrates for phosphorylation, the co-active kinase ROP17 targets adjacent switch-I threonines, and dense granule proteins GRA7 and GRA60 act in the same IRG-resistance pathway [#9, #10, #12, #13, #31]. ROP18 kinase activity is regulated by autophosphorylation of its N-terminal extension, which relieves autoinhibition, and a second ligand-binding pocket outside the active site independently contributes to virulence [#2, #11]. Beyond IRG evasion, ROP18 phosphorylates and destabilizes host targets including ATF6\\u03b2 to suppress CD8 T-cell responses, and phosphorylates Smad1, RTN1-C, and vimentin, linking it to modulation of host signalling, ER stress, and apoptosis [#5, #21, #23, #24]. Allelic and expression polymorphism\\u2014including loss of an upstream regulatory element in the type III lineage\\u2014accounts for much of the strain-dependent variation in virulence [#17].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that a single secreted parasite kinase can determine acute virulence, defining ROP18 as a genetically mapped effector rather than a passive antigen.\",\n      \"evidence\": \"QTL mapping and positional cloning with transfection of the virulent allele into a nonpathogenic type III strain and kinase-dead mutant analysis in a mouse virulence assay\",\n      \"pmids\": [\"17170305\", \"17170306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host substrate(s) not yet identified\", \"Mechanism connecting kinase activity to virulence unknown\", \"Subcellular site of action not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I); type III\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Confirmed ROP18 is an intrinsically active serine-threonine kinase and localized it to rhoptries with re-targeting to the PVM upon invasion, placing the enzyme at the host-parasite interface.\",\n      \"evidence\": \"In vitro kinase assay with recombinant catalytic domain, transgenic overexpression with kinase-dead control, and immunofluorescence/fractionation in RH\",\n      \"pmids\": [\"17305424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the ~70 kDa substrate not established\", \"Mechanism of PVM re-localization unknown\", \"No host substrate identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined how the kinase is switched on, showing autophosphorylation of the N-terminal extension relieves autoinhibition of the ATP-binding pocket.\",\n      \"evidence\": \"X-ray structures of ROP2/ROP8 pseudokinases used for homology modelling plus autophosphorylation assays, mutagenesis, and mass spectrometry\",\n      \"pmids\": [\"19197235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Autoinhibition model later challenged by direct ROP18 kinase structure\", \"Physiological ROP2/ROP8 phosphorylation significance unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Explained natural virulence variation by linking three ROP18 alleles and an upstream regulatory element to expression differences, showing loss of the element silenced ROP18 in the ancient type III lineage.\",\n      \"evidence\": \"Population sequencing of natural isolates, promoter deletion, expression measurement, and virulence assays across strains\",\n      \"pmids\": [\"19266027\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of allelic activity differences not fully resolved\", \"Regulatory element trans-factors unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III; diverse natural isolates\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified the key host substrate class, showing ROP18 phosphorylates IRGs (Irga6) on switch-I threonines to inactivate them and block PVM recruitment, defining the mechanism of macrophage evasion.\",\n      \"evidence\": \"Co-IP, in vitro phosphorylation with substrate profiling, \\u0394rop18 knockout, IFN-\\u03b3 macrophage clearance and mouse virulence assays; phospho-site mapping and GTPase activity readouts\",\n      \"pmids\": [\"21147463\", \"21203588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full IRG substrate range not yet delimited\", \"Requirement for co-factors not yet appreciated\", \"Human-cell relevance unaddressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended ROP18's targets beyond IRGs to the host ER transcription factor ATF6\\u03b2, linking the kinase to suppression of adaptive (CD8 T-cell) immunity.\",\n      \"evidence\": \"Reciprocal Co-IP, mammalian overexpression, \\u0394rop18, proteasome inhibition, and ATF6\\u03b2-KO mouse challenge in RH\",\n      \"pmids\": [\"21670204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phospho-site on ATF6\\u03b2 not mapped\", \"E3 ligase mediating degradation unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed ROP18 genetically upstream of GBP-mediated killing and revealed its dependence on ROP5, showing both are required to prevent Gbp1 recruitment.\",\n      \"evidence\": \"\\u0394rop18/\\u0394rop5 knockouts, Gbp1\\u2212/\\u2212 macrophages and mice, systematic ROP16/ROP18/GRA15 knockout comparison with confocal imaging\",\n      \"pmids\": [\"23633952\", \"21931713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect effect on GBPs vs IRGs not separated\", \"Mechanism of ROP5 dependence not yet defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the co-factor requirement, showing ROP5 pseudokinases directly regulate ROP18 catalytic activity and allosterically prime IRG substrates, and that PVM tethering via the arginine-rich N-terminus is essential for function.\",\n      \"evidence\": \"Allelic swaps, structural/biochemical IRG binding and oligomerization assays, in vitro kinase activity assays, N-terminal deletion mutagenesis with virulence readouts, and human vs mouse cell comparison\",\n      \"pmids\": [\"22761577\", \"22802726\", \"23144612\", \"22906355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the ROP5\\u2013ROP18\\u2013IRG ternary arrangement incomplete\", \"Human-cell IRG-independent function undefined at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II; type III\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Refined the activation model by determining the ROP18 kinase-domain crystal structure, which contradicted the earlier autoinhibition model and revealed a second ligand-binding pocket required for virulence.\",\n      \"evidence\": \"X-ray crystallography with sucrose-bound pocket, mutagenesis of the second pocket, and mouse virulence assays in RH\",\n      \"pmids\": [\"24129568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological ligand of the second pocket unknown\", \"Reconciliation with N-terminal autophosphorylation model incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated cross-species conservation and regulatory-element origin, showing H. hammondi ROP18 complements T. gondii and that the promoter element silenced in type III was ancestrally active.\",\n      \"evidence\": \"Genomic sequencing, cross-species complementation in T. gondii, promoter deletion, and virulence assays\",\n      \"pmids\": [\"23589877\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional divergence of H. hammondi complex components unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type III\"}, {\"species\": \"Hammondia hammondi\", \"strain\": \"HhCatGer041\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the full PVM virulence complex, showing ROP18 associates with ROP5, ROP17, ROP2/ROP8, and GRA7, and that ROP17 and ROP18 cooperatively phosphorylate adjacent switch-I threonines.\",\n      \"evidence\": \"Tandem affinity purification-MS in T. gondii, Co-IP, in vitro kinase assays, single and double knockouts, IRG polymerization and recruitment assays in RH\",\n      \"pmids\": [\"24390541\", \"24832449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the complex not resolved\", \"Division of labor among kinases only partially defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed via heterologous gain-of-function that ROP18 kinase activity is the decisive functional difference distinguishing virulent T. gondii from related parasites carrying a ROP18 pseudogene.\",\n      \"evidence\": \"Transgenic TgROP18 expression in Neospora caninum with mouse virulence and IRG phosphorylation assays\",\n      \"pmids\": [\"24927100\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether N. caninum provides all cognate co-factors unclear\"],\n      \"attribution\": [{\"species\": \"Neospora caninum\", \"strain\": \"Nc1\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established Irga6 as the specific ROP18 target and clarified GRA7's role, showing GRA7 tethers to the PVM via ROP5 and enhances Irga6 phosphorylation.\",\n      \"evidence\": \"Co-IP (GRA7\\u2013ROP5), in vitro phosphorylation against a panel of IRG proteins, knockouts, and mouse virulence assays in RH\",\n      \"pmids\": [\"26247512\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why other IRGs are not phosphorylated not fully explained mechanistically\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Broadened the evolutionary significance of ROP18 by showing complete virulence attenuation upon deletion across divergent South American strains, contrasting with partial attenuation in RH.\",\n      \"evidence\": \"CRISPR-Cas9 deletion and complementation in type 4, 8, and 10 strains with mouse virulence assays\",\n      \"pmids\": [\"26291965\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Strain-specific co-factor differences underlying the phenotype not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"VAND (type 10); TgCtBr5 (type 8); TgCtBr18 (type 4)\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Uncovered a kinase-independent, PVM-localization-dependent role for ROP18 in host manipulation, distinct from its IRG-phosphorylating function.\",\n      \"evidence\": \"\\u0394rop18 and PVM-localization mutants in an antitumor immunity mouse ovarian cancer model\",\n      \"pmids\": [\"27447180\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular effector of the kinase-independent function unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I uracil auxotroph (\\u0394ku80)\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended ROP18's essentiality to chronic infection, showing kinase activity and PVM association are required for IRG resistance and cyst formation in type II.\",\n      \"evidence\": \"Systematic ROPK-locus deletions, kinase-dead and localization mutants, and cyst-burden/virulence assays in ME49 (type II)\",\n      \"pmids\": [\"27165797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Type II vs type I mechanistic differences in cyst context not fully resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49 (type II)\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Systematically expanded the host substrate repertoire via proteome-array screening, identifying Smad1 (Ser-187), p53, p38, and UBE2N and vimentin as ROP18 targets linked to degradation and invasion control.\",\n      \"evidence\": \"Human proteome array probed with recombinant ROP18, in vitro kinase assays, phospho-site mutagenesis, proteasome inhibition, and vimentin-KO infection assays in RH\",\n      \"pmids\": [\"28087594\", \"29104504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which substrates are phosphorylated in vivo during infection not established\", \"Physiological consequences of most targets not fully mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected ROP18 to CNS pathology by identifying RTN1-C as a phospho-substrate whose modification triggers ER-stress apoptosis in neural cells.\",\n      \"evidence\": \"Proteome-array pull-down, Co-IP, in vitro phosphorylation with site mapping, ER-stress markers, and HDAC activity assays in RH\",\n      \"pmids\": [\"29784816\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance to encephalitis not directly established\", \"Link between phosphorylation and GRP78 acetylation mechanistically incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a cell-type-specific anti-apoptotic function, showing ROP18 interacts with the ATP-gated channel P2X1 to block mitochondrial apoptosis.\",\n      \"evidence\": \"FRET, Co-IP, flow cytometry for apoptosis/Ca2+ influx, and apoptosis-marker western blotting in P2X1-expressing SF268 cells in RH\",\n      \"pmids\": [\"31164145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether interaction requires kinase activity unclear\", \"In vivo relevance not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Added GRA60 to the IRG-resistance pathway, showing it associates with ROP18 and its loss phenocopies \\u0394rop5 for IRG recruitment.\",\n      \"evidence\": \"Co-IP, single and double knockouts, IRG (Irgb10/Irga6) recruitment confocal assays, and virulence assays in RH\",\n      \"pmids\": [\"33040458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical role of GRA60 in the complex undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined ROP18-mediated evasion in human cells by targeting TRIM21 for lysosomal degradation, reducing PVM ubiquitination and parasite killing.\",\n      \"evidence\": \"Co-IP, degradation and lysosomal-inhibitor assays, TRIM21 knockdown/overexpression, and PVM ubiquitination confocal microscopy for TgROP18I in RH\",\n      \"pmids\": [\"34124071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation of TRIM21 not conclusively mapped\", \"Relationship to IRG-independent human resistance incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed ROP18 suppresses type I interferon and autophagic PVM targeting by interacting with IRF3, p62, and TRAF6 in both murine and human cells.\",\n      \"evidence\": \"Co-IP, IFN-\\u03b2 reporter assay, p62 phosphorylation and PVM ubiquitin/LC3 western blotting and confocal microscopy for TgROP18I\",\n      \"pmids\": [\"35084749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether these interactions require kinase activity not resolved\", \"In vivo contribution not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked ROP18 to reproductive pathology, showing it lowers CD73 on decidual macrophages via LSD1/SNAIL1 signalling to impair maternal-fetal immune tolerance.\",\n      \"evidence\": \"RH-\\u0394rop18 infection of pregnant mice and human decidual tissue, flow cytometry, western blotting, and ChIP assays in RH\",\n      \"pmids\": [\"39994736\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the effect is direct or downstream of general infection burden not fully separated\", \"Kinase-dependence not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ROP18's many reported host substrates and interactions (Smad1, p53, p38, RTN1-C, vimentin, TRIM21, IRF3, P2X1) are prioritized and coordinated in vivo, and how kinase-dependent versus kinase-independent PVM functions are integrated, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model separating in-vivo-relevant substrates from proteome-array hits\", \"Structural basis reconciling autophosphorylation activation with the crystal-structure autoinhibition rebuttal not settled\", \"Kinase-independent PVM effector remains unidentified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004674\", \"supporting_discovery_ids\": [0, 1, 3, 4, 24], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]},\n      {\"term_id\": \"GO:0106310\", \"supporting_discovery_ids\": [3, 4, 15, 21, 23, 24], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]},\n      {\"term_id\": \"GO:0046777\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005524\", \"supporting_discovery_ids\": [2, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]},\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [1, 7, 12, 14], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051607\", \"supporting_discovery_ids\": [3, 4, 6, 8, 15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]},\n      {\"term_id\": \"GO:0050690\", \"supporting_discovery_ids\": [5, 25, 26], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]},\n      {\"term_id\": \"GO:0043066\", \"supporting_discovery_ids\": [22], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]},\n      {\"term_id\": \"GO:0030178\", \"supporting_discovery_ids\": [28], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}]}\n    ],\n    \"complexes\": [\n      \"ROP18\\u2013ROP5 pseudokinase\\u2013ROP17\\u2013GRA7 PVM virulence complex\",\n      \"ROP18\\u2013ROP2/ROP8\\u2013GRA7 PVM complex\",\n      \"ROP18\\u2013GRA60 IRG-resistance association\"\n    ],\n    \"partners\": [\n      \"ROP5 (pseudokinase co-factor and catalytic regulator)\",\n      \"ROP17 (co-active kinase, adjacent switch-I threonines)\",\n      \"GRA7 (dense granule protein, Irga6 binding)\",\n      \"GRA60 (dense granule protein, IRG-resistance pathway)\",\n      \"Irga6 / immunity-related GTPases (substrate)\",\n      \"ATF6\\u03b2 (host, degraded)\",\n      \"Smad1 / p53 / p38 / UBE2N (host substrates)\",\n      \"RTN1-C (host substrate)\",\n      \"vimentin (host substrate)\",\n      \"TRIM21 (host, degraded)\",\n      \"IRF3, p62, TRAF6 (host signalling)\",\n      \"P2X1 (host ion channel)\"\n    ],\n    \"other_free_text\": [\n      \"Activation involves autophosphorylation of the N-terminal extension relieving autoinhibition (#2); a later crystal structure disputes this autoinhibition model and defines a second ligand-binding pocket required for virulence (#11).\",\n      \"PVM tethering is mediated by an arginine-rich N-terminal domain and is essential for both kinase-dependent IRG evasion and a kinase-independent host-manipulation function (#7, #27).\",\n      \"Polymorphism: three principal alleles and an upstream regulatory element govern expression and virulence; type III lost the element and ROP18 expression (#17, #19).\",\n      \"PMID:24648522 (NF-\\u03baB/p65 degradation) was withdrawn and is excluded from evidence (#30).\"\n    ]\n  },\n  \"strain_conflicts\": [\n    {\n      \"aspect\": \"Virulence dependence on ROP18 (attenuation upon deletion)\",\n      \"claims\": [\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH (type I)\"}], \"claim\": \"\\u0394rop18 causes only partial attenuation of acute virulence in type I RH\", \"supporting_discovery_ids\": [16]},\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"VAND (type 10); TgCtBr5 (type 8); TgCtBr18 (type 4)\"}], \"claim\": \"Deletion of ROP18 causes complete attenuation of virulence in South American type 4, 8, and 10 strains\", \"supporting_discovery_ids\": [16]},\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49 (type II)\"}], \"claim\": \"ROP18 kinase activity and PVM association are required for IRG resistance and chronic cyst formation in type II\", \"supporting_discovery_ids\": [14]}\n      ]\n    },\n    {\n      \"aspect\": \"ROP18 expression across natural alleles\",\n      \"claims\": [\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I; type II\"}], \"claim\": \"ROP18 is expressed and confers virulence, with allele-dependent activity\", \"supporting_discovery_ids\": [17, 4]},\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type III\"}], \"claim\": \"Loss of an upstream regulatory element eliminated ROP18 expression and virulence\", \"supporting_discovery_ids\": [17, 19]}\n      ]\n    },\n    {\n      \"aspect\": \"Irga6 phosphorylation efficiency by ROP18 allele\",\n      \"claims\": [\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}], \"claim\": \"The virulent type I allele efficiently phosphorylates mouse Irga6 at two switch-I threonines\", \"supporting_discovery_ids\": [4]},\n        {\"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II; type III\"}], \"claim\": \"Phosphorylation of Irga6 is markedly weaker (type I > type II/III), correlating with lower virulence\", \"supporting_discovery_ids\": [4]}\n      ]\n    }\n  ]\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_205250","name":"ROP18","product":"rhoptry protein ROP18","uniprot_acc":"A0A125YQL1","chromosome":"TGME49_chrVIIa","start_pos":1513497},{"strain":"GT1","gene_id":"TGGT1_205250","name":null,"product":"rhoptry protein ROP18","uniprot_acc":null,"chromosome":"TGGT1_chrVIIa","start_pos":1426782},{"strain":"RH88","gene_id":"TGRH88_034260","name":null,"product":"rhoptry protein ROP18","uniprot_acc":null,"chromosome":"CM023088","start_pos":1595886},{"strain":"VEG","gene_id":"TGVEG_205250","name":null,"product":"rhoptry protein ROP18","uniprot_acc":"V4ZDA0","chromosome":"TGVEG_chrVIIa","start_pos":1584812}]}