{"gene":"TG_254470","gene_symbol":"MYR1","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2016,"finding":"MYR1 (TGGT1_254470) is a secreted protein that is cleaved by TgASP5 into two stable fragments, both of which localize within the parasitophorous vacuole (PV) and at the parasitophorous vacuole membrane (PVM). Deletion of MYR1 abolishes GRA24 transit to the host nucleus, establishing MYR1 as essential machinery for translocation of dense granule effectors (including GRA16 and GRA24) from the PV into the host cell cytosol and nucleus. MYR1 is also required for host c-Myc upregulation.","method":"Whole-genome sequencing of flow-cytometry-selected mutants; immunofluorescence localization; genetic deletion (ΔMyR1) with host c-Myc and GRA24 nuclear transit as phenotypic readouts; TgASP5-dependent cleavage assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and cell-biological approaches, multiple orthogonal readouts (c-Myc induction, GRA24 nuclear localization, GRA16 function), foundational paper replicated by multiple subsequent studies","pmids":["26838724"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"finding":"MYR1 forms a protein complex at the PVM with MYR2 and MYR3. MYR2 and MYR3 are secreted into the PV space and co-localize with PVM and MYR1. MYR3, but not MYR2, stably associates with MYR1. The N- and C-terminal fragments of MYR1 are disulfide-linked. All three proteins are membrane-associated (consistent with predicted transmembrane domains). MYR2 and MYR3 are required for translocation of a subset of GRAs (GRA16, GRA24) into the host cell.","method":"Co-immunoprecipitation; immunofluorescence co-localization; genetic screens (c-Myc and GRA export assays in mutants); DHFR domain fusion block-of-function assay supporting a translocon model","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal methods (co-localization, genetic epistasis, domain-fusion block), replicated by subsequent MYR complex studies","pmids":["29357375"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"finding":"RNA-Seq of human foreskin fibroblasts infected with RHΔmyr1 vs. wild-type established that the majority of differentially regulated host genes responding to Toxoplasma infection are MYR1-dependent. A subset of MYR1-dependent responses are ASP5-independent, indicating MYR1 function does not strictly require ASP5 cleavage for export of some effectors. MYR1-dependent effects include upregulation of E2F transcription factors/cell cycle genes and downregulation of interferon signaling.","method":"RNA-Seq transcriptomics of infected host cells; genetic deletion of MYR1 and ASP5 compared to wild-type","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined transcriptome readout, single lab, single primary method (RNA-Seq)","pmids":["29615509"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"finding":"ROP17, a rhoptry serine/threonine protein kinase, is required for MYR1-dependent translocation of dense granule effectors across the PVM. Null or catalytically dead ROP17 mutants are defective in GRA translocation. ROP17 acts from the host cytosolic side of the PVM (trans-rescue by ROP17 from co-infecting parasites rescues translocation), placing it as a component upstream of or parallel to the MYR translocon complex.","method":"Whole-genome sequencing of translocation-defective mutants; genetic complementation and trans-rescue experiments; catalytic dead mutant construction; immunofluorescence","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Strong — catalytically dead mutant, trans-rescue epistasis, multiple genetic and cell-biological approaches in single study","pmids":["31366709"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"finding":"HCE1, a novel dense granule effector, is exported from the PV in a MYR1-dependent manner and localizes to the host nucleus, where it binds E2F3, E2F4, and DP1 transcription factors (shown by immunoprecipitation from infected host cells) and is necessary and sufficient to upregulate cyclin E and E2F target genes.","method":"Genetic deletion (ΔHce1); immunoprecipitation from infected host cells; expression in uninfected HFFs and Neospora caninum for sufficiency; immunofluorescence","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined nuclear phenotype, Co-IP identifying binding partners, heterologous expression for sufficiency, multiple orthogonal methods","pmids":["31040242"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"Affinity purification of MYR1 identified interacting proteins MYR4 (TGGT1_211460), GRA54 (TGGT1_204340), PPM3C (TGGT1_270320), GRA44, and GRA45 within the PV. Of these, GRA44, GRA45, and MYR4 are each essential for GRA16 translocation and host c-Myc/cyclin E1 upregulation, expanding the translocon complex to at least eight discrete proteins.","method":"Affinity purification/Co-immunoprecipitation of MYR1 followed by mass spectrometry; genetic deletion of identified interactors with GRA16 translocation and c-Myc/cyclin E1 as functional readouts","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Strong — AP-MS identification of interactors confirmed by genetic deletion with multiple functional readouts, rigorous mechanistic follow-up","pmids":["32075880"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"GRA44, which contains a putative acid phosphatase domain, physically interacts with MYR1 (shown by immunoprecipitation) and is required for host cell c-Myc upregulation downstream of effector secretion, phenocopying loss of the MYR translocon.","method":"Immunoprecipitation; conditional knockdown of GRA44; c-Myc upregulation assay","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus conditional KD with functional readout, single lab, moderate replication (consistent with PMID:32075880)","pmids":["32075881"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"MYR1 is identified as a component of the cyst wall proteome in vitro. During tachyzoite-to-bradyzoite transition, the export of effectors (GRA16, GRA24, TgIST, and others) into the host nucleus occurs primarily during early time points post-infection, with diminishing nuclear accumulation at later stages. TgIST-mediated suppression of IFN-γ signaling persists in bradyzoite infection, demonstrating continued functional effector export beyond early stages.","method":"Epitope tagging of endogenous effector loci; immunofluorescence assays at multiple time points during bradyzoite differentiation; quantification of host nuclear accumulation","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct imaging with epitope-tagged endogenous loci at multiple time points, single lab, IFN-γ suppression functional readout","pmids":["32156805"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"TgPPM3C, a PP2C-class serine/threonine phosphatase that co-immunoprecipitates with MYR1, is required for export of GRA16 and GRA28 but not other MYR1-dependent effectors. Phosphoproteomic analysis shows that loss of PPM3C alters phosphorylation of GRA16, GRA28, and MYR1 itself. Phosphomimetic mutation of two GRA16 serine residues causes export defects, indicating that de-phosphorylation by PPM3C is a prerequisite for GRA16 export through the MYR translocon.","method":"Genetic deletion of PPM3C; phosphoproteomics; phosphomimetic mutagenesis of GRA16; immunofluorescence for effector nuclear localization; prior Co-IP establishing MYR1 interaction (PMID:32075880)","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — combines phosphoproteomic analysis, phosphomimetic mutagenesis, and genetic KO with defined export phenotype; multiple orthogonal methods in single study","pmids":["33370417"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"Single-cell transcriptomic analysis of uninfected-injected (U-I) cells versus infected cells from wild-type and ΔMyR1 parasites established that the host's earliest transcriptional response to infection is driven primarily by injected ROPs, not MYR1-dependent GRAs. MYR1-dependent GRA effectors counter-act ROP-driven pro-inflammatory signatures, and MYR1-dependent paracrine effects further modulate inflammatory responses in bystander cells.","method":"Single-cell RNA-Seq of flow-sorted U-I, infected, and uninfected cells; genetic deletion of MYR1","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — scRNA-Seq with ΔMyR1 comparison, novel U-I cell enrichment method, single lab","pmids":["32518180"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2021,"finding":"In bradyzoites, MYR1-dependent protein export is responsible for transcriptional remodeling of host cells, including suppression of type I IFN and IFN-γ signaling pathways. Loss of MYR1 abrogates this transcriptional remodeling and removes protection of host cells from IFN-γ-mediated cell death.","method":"Genetic deletion of MYR1 in bradyzoite stage; RNA-Seq; IFN-γ-mediated cell death assays","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with RNA-Seq and functional cell death readout, demonstrates MYR1-dependent export is active and functionally essential in bradyzoite stage","pmids":["34921775"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"GRA28, a dense granule effector, is released from the PV via the MYR1 secretory pathway, traffics to the host cell nucleus, and cooperates with host chromatin remodelers to drive CCR7 expression and chemotactic migration of parasitized macrophages. In vivo, MYR1-deficient parasites are outcompeted in dissemination by wild-type parasites.","method":"Genetic deletion of MYR1 and GRA28; adoptive transfer in vivo migration assays; immunofluorescence; chromatin remodeler interaction studies","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic deletion of MYR1 and GRA28 with in vitro and in vivo migration phenotype, mechanistic link to chromatin remodeling, multiple orthogonal methods","pmids":["36309013"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"ASP5 is required for proteolytic cleavage of MYR1 and other substrates (GRAs) at an ASPH motif in the Golgi, enabling their proper localization within the PV/PVM and their function in the MYR1/2/3 export translocon. Bradyzoite-stage ASP5 substrates also participate in cyst wall formation.","method":"N-terminal amine isotopic labeling of substrates (TAILS) to map cleavage sites; genetic deletion of ASP5; comparison of tachyzoite and bradyzoite N-terminomes","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — TAILS N-terminome proteomics plus genetic KO, single lab, provides direct identification of ASP5 cleavage sites on MYR1 pathway components","pmids":["36210525"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"MYR1 is required for T. gondii-induced early S-phase arrest and double-strand DNA breaks in primary human host cells within 15 min of infection. ΔMyR1 tachyzoites fail to induce S-phase arrest and DNA damage foci. This MYR1-dependent DNA damage is ROS-independent and is not mediated by HCE1/cyclin E1 overexpression.","method":"Genetic deletion of MYR1 and HCE1; cell cycle analysis; γH2AX foci quantification for DNA damage; ROS inhibitor experiments; time-course immunofluorescence","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple cell-biology readouts (cell cycle, DNA damage markers), ROS exclusion controls, single lab","pmids":["39681694"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"finding":"MYR1-dependent secreted proteins generate a permissive host niche in a paracrine manner: wild-type parasites co-infecting the same host can rescue ΔMYR1 parasite growth in vivo, independent of adaptive immunity or key pro-inflammatory cytokines. MYR1 is not required for parasite survival within IFN-γ-activated macrophages, indicating its primary function is paracrine niche creation rather than protection of the infected cell.","method":"Co-infection experiments in mice with WT and ΔMYR1 parasites; immunodeficient mouse models; cytokine knockout mouse models; in vitro macrophage survival assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple in vivo and in vitro genetic experiments with multiple host immune pathway knockouts, rigorous trans-rescue epistasis, replicates prior CRISPR screen observation (PMID:31481656)","pmids":["39654402"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2026,"finding":"Activation of the non-canonical NF-κB pathway (nuclear accumulation of RelB and p52) during T. gondii infection is strictly MYR1-dependent. Mechanistically, MYR1-dependent effectors drive depletion of the negative regulator TRAF3, leading to NIK stabilization, p100 phosphorylation, and p52 processing. Eight MYR1-dependent effectors (IST, NSM, HCE1/TEEGR, GRA16, GRA18, GRA24, GRA28, GRA84) function additively through this pathway; no single effector is solely responsible.","method":"Genetic deletion of MYR1; combinatorial knockout parasites lacking multiple effectors; immunoblot for TRAF3/NIK/p100/p52; immunofluorescence for RelB/p52 nuclear accumulation","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with mechanistic downstream readouts, combinatorial KO panel, preprint not yet peer-reviewed","pmids":["41959509"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]}],"current_model":"MYR1 is a secreted, TgASP5-cleaved protein that localizes to the parasitophorous vacuole membrane (PVM) where it forms the core of a multi-protein translocon complex (with MYR2, MYR3, MYR4, GRA44, GRA45, ROP17, and PPM3C, among others) that is essential for translocation of dense granule effector proteins from the PV lumen across the PVM into the host cell cytosol and nucleus, thereby enabling Toxoplasma to reprogram host transcription (c-Myc, cyclin E, E2F targets, IFN signaling, non-canonical NF-κB), induce early host DNA damage and cell-cycle arrest, drive macrophage chemotactic dissemination, and create a permissive paracrine niche in vivo — all of which are required for full parasite virulence."},"narrative":{"mechanistic_narrative":"MYR1 is a secreted Toxoplasma gondii protein that forms the core of a parasitophorous vacuole membrane (PVM) translocon required for exporting dense granule (GRA) effector proteins from the vacuolar lumen into the host cell cytosol and nucleus, thereby enabling the parasite to reprogram host transcription [PMID:26838724, PMID:29357375]. MYR1 is processed by the Golgi aspartyl protease TgASP5 into two disulfide-linked, stably associated fragments that localize to the PV and PVM [PMID:26838724, PMID:36210525]. At the PVM it assembles with MYR2, MYR3, and MYR4 and additional interactors GRA44, GRA45, GRA54, and the PP2C phosphatase PPM3C into a multi-protein complex, with MYR3, MYR4, GRA44, and GRA45 each individually required for effector translocation and downstream host c-Myc/cyclin E1 induction [PMID:29357375, PMID:32075880, PMID:32075881]. Translocation additionally depends on the rhoptry kinase ROP17 acting from the host-cytosolic face of the PVM, and on PPM3C-mediated dephosphorylation of specific GRA16 serines as a prerequisite for that effector's export [PMID:31366709, PMID:33370417]. Through this pathway MYR1 delivers effectors including GRA16, GRA24, HCE1/TEEGR, GRA28, and TgIST that collectively drive the majority of the host transcriptional response to infection — upregulating E2F/cell-cycle genes, suppressing type I and type II interferon signaling, activating the non-canonical NF-κB pathway via TRAF3 depletion, inducing early S-phase arrest and DNA double-strand breaks, and driving CCR7-dependent chemotactic dissemination of parasitized macrophages [PMID:29615509, PMID:31040242, PMID:34921775, PMID:36309013, PMID:39681694, PMID:41959509]. In vivo, MYR1-dependent secretion creates a permissive paracrine host niche that supports parasite growth independent of adaptive immunity, establishing this export machinery as central to virulence [PMID:39654402]. MYR1-dependent effector export persists and remains functionally essential in the bradyzoite stage [PMID:34921775].","teleology":[{"year":2016,"claim":"Established that Toxoplasma requires a dedicated machinery to move dense granule effectors across the PVM into the host, answering how vacuole-confined parasites reach the host nucleus.","evidence":"Whole-genome sequencing of translocation-defective mutants, genetic deletion of MYR1, and TgASP5-dependent cleavage assays with GRA24 nuclear transit and host c-Myc as readouts in RH strain","pmids":["26838724"],"confidence":"High","gaps":["Did not resolve whether MYR1 is a channel, chaperone, or scaffold","Additional complex components unknown","Molecular mechanism of substrate recognition not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"claim":"Defined MYR1 as part of a membrane-associated PVM complex, showing the translocon is multi-subunit rather than a single protein.","evidence":"Co-immunoprecipitation, immunofluorescence co-localization, genetic epistasis, and DHFR domain-fusion block-of-function assays identifying MYR2/MYR3 in RH strain","pmids":["29357375"],"confidence":"High","gaps":["Stoichiometry and architecture of the complex unresolved","MYR2 association mode with MYR1 unclear (does not stably bind)","Mechanism of substrate passage not shown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"claim":"Quantified the scope of MYR1 control over the host response, showing most infection-induced host gene changes depend on MYR1 and that some effects are ASP5-independent.","evidence":"RNA-Seq of infected human fibroblasts comparing wild-type, Δmyr1, and Δasp5 in RH strain","pmids":["29615509"],"confidence":"Medium","gaps":["Individual effectors responsible for each transcriptional signature not mapped","Mechanism of ASP5-independent export undefined","Single-method (RNA-Seq) study"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"claim":"Identified a kinase requirement acting from the host side of the PVM, revealing translocation depends on phosphoregulation across the membrane.","evidence":"Whole-genome sequencing of mutants, catalytically-dead ROP17 mutants, and trans-rescue complementation in RH strain","pmids":["31366709"],"confidence":"High","gaps":["ROP17 substrates in the translocation process not identified","Whether ROP17 acts on the translocon or on effectors unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"claim":"Linked a specific MYR1-exported effector to host cell-cycle reprogramming, giving mechanistic identity to the E2F/cyclin E transcriptional signature.","evidence":"Genetic deletion of HCE1, Co-IP identifying E2F3/E2F4/DP1 binding, and heterologous expression for sufficiency in RH strain","pmids":["31040242"],"confidence":"High","gaps":["Structural basis of HCE1–E2F/DP1 interaction unknown","Does not explain the DNA-damage arm of the response"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"Expanded the translocon to at least eight subunits by unbiased purification, defining the full complex needed for export.","evidence":"Affinity purification/Co-IP of MYR1 with mass spectrometry plus genetic deletion of MYR4/GRA44/GRA45 with GRA16 translocation and c-Myc/cyclin E1 readouts in RH strain","pmids":["32075880","32075881"],"confidence":"High","gaps":["Roles of GRA54 not functionally defined","Enzymatic activity of GRA44 acid phosphatase domain not tested directly","Complex assembly order unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"Showed that dephosphorylation is a gating step for export, revealing PPM3C-mediated GRA16 dephosphorylation is a prerequisite for translocation of a subset of effectors.","evidence":"Genetic deletion of PPM3C, phosphoproteomics, and phosphomimetic mutagenesis of GRA16 serines with nuclear-localization readouts","pmids":["33370417"],"confidence":"High","gaps":["Why only a subset of effectors (GRA16, GRA28) require PPM3C is unclear","Kinase counteracting PPM3C not identified","Functional consequence of MYR1 phosphorylation change not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Resolved the temporal hierarchy of host manipulation, showing injected ROPs drive the earliest response while MYR1-dependent GRAs counteract ROP-driven inflammation, including in bystander cells.","evidence":"Single-cell RNA-Seq of flow-sorted uninfected-injected, infected, and uninfected cells with Δmyr1 comparison in RH strain","pmids":["32518180"],"confidence":"Medium","gaps":["Effectors mediating paracrine effects not identified","Single-lab scRNA-Seq"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"Placed MYR1 in the ASP5 substrate map and cyst wall proteome, and showed effector export operates through bradyzoite differentiation.","evidence":"TAILS N-terminomics with ASP5 KO, cyst wall proteomics, and time-course imaging of epitope-tagged effectors during bradyzoite transition","pmids":["32156805","36210525"],"confidence":"Medium","gaps":["Functional role of MYR1 in the cyst wall vs. translocation not separated","Kinetics of complex turnover across stages unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"Demonstrated MYR1-dependent export is functionally essential in the persistent bradyzoite stage, protecting host cells from IFN-γ-mediated death.","evidence":"Genetic deletion of MYR1 in bradyzoites, RNA-Seq, and IFN-γ-mediated cell death assays","pmids":["34921775"],"confidence":"High","gaps":["Individual effectors driving bradyzoite-stage protection not fully mapped","In vivo relevance during chronic infection not addressed here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"Connected MYR1-dependent effector export to organismal dissemination, showing GRA28 drives CCR7-dependent macrophage migration and MYR1 loss impairs in vivo spread.","evidence":"Genetic deletion of MYR1 and GRA28, chromatin-remodeler interaction studies, and adoptive-transfer in vivo migration assays","pmids":["36309013"],"confidence":"High","gaps":["Chromatin remodeler identities and mechanism at the CCR7 locus not fully defined","Contribution of migration vs. other MYR1 functions to dissemination not dissected"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Uncovered a rapid MYR1-dependent host DNA-damage and cell-cycle arrest program independent of the HCE1/cyclin E axis, showing MYR1 controls damage responses beyond transcriptional reprogramming.","evidence":"Genetic deletion of MYR1 and HCE1, γH2AX foci quantification, cell-cycle analysis, and ROS-inhibitor controls in RH strain","pmids":["39681694"],"confidence":"Medium","gaps":["Effector responsible for DNA damage not identified","Molecular trigger of double-strand breaks unknown","Single-lab study"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"claim":"Defined the ultimate in vivo purpose of MYR1 secretion as paracrine niche creation rather than protection of the infected cell.","evidence":"WT/ΔMYR1 co-infection trans-rescue in mice, immunodeficient and cytokine-knockout models, and in vitro macrophage survival assays in RH strain","pmids":["39654402"],"confidence":"High","gaps":["Secreted factors mediating the paracrine niche not identified","Host receptors/pathways receiving the paracrine signal unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2026,"claim":"Assigned MYR1-dependent effectors to non-canonical NF-κB activation, showing eight effectors act additively to deplete TRAF3 and process p52.","evidence":"Genetic deletion of MYR1, combinatorial multi-effector knockout parasites, and immunoblot/immunofluorescence for TRAF3/NIK/p100/p52 and RelB/p52 nuclear accumulation in RH and ME49 (preprint)","pmids":["41959509"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Direct molecular mechanism by which each effector depletes TRAF3 not resolved","Relative contribution of individual effectors not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":null,"claim":"The biophysical mechanism of substrate passage across the PVM — how the MYR translocon recognizes, unfolds, and threads effectors — remains undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the translocon","Channel vs. chaperone mechanism unresolved","Energetics and driving force of translocation unknown"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MYR translocon complex at the PVM: MYR1, MYR2, MYR3, MYR4, GRA44, GRA45, GRA54, PPM3C [#1, #5]"],"partners":["MYR3 (STABLE ASSOCIATION WITH MYR1) [#1]","MYR2 [#1]","MYR4 [#5]","GRA44 (PUTATIVE ACID PHOSPHATASE DOMAIN) [#5, #6]","GRA45 [#5]","GRA54 [#5]","PPM3C (PP2C-CLASS PHOSPHATASE) [#5, #8]","ROP17 (RHOPTRY KINASE, ACTS FROM HOST-CYTOSOLIC FACE) [#3]","TGASP5 (PROTEASE THAT CLEAVES MYR1) [#0, #12]"],"other_free_text":["MYR1 is cleaved by TgASP5 into two disulfide-linked fragments that both localize to the PV/PVM [#0, #1, #12].","MYR1-dependent exported effectors include GRA16, GRA24, HCE1/TEEGR, GRA28, TgIST, GRA18, NSM, GRA84 [#0, #4, #8, #11, #15].","PPM3C-mediated dephosphorylation of specific GRA16 serines is a prerequisite for export of a subset of effectors [#8].","MYR1-dependent host outcomes: c-Myc/cyclin E induction, E2F target upregulation, interferon suppression, non-canonical NF-κB activation via TRAF3 depletion, early S-phase arrest and DNA double-strand breaks, CCR7-driven macrophage chemotaxis, and paracrine niche creation [#0, #2, #4, #10, #11, #13, #14, #15]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"26838724","id":"PMC_26838724","title":"A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells.","date":"2016","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/26838724","citation_count":133,"is_preprint":false},{"pmid":"31481656","id":"PMC_31481656","title":"A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31481656","citation_count":76,"is_preprint":false},{"pmid":"29357375","id":"PMC_29357375","title":"Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii.","date":"2018","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/29357375","citation_count":75,"is_preprint":false},{"pmid":"32075880","id":"PMC_32075880","title":"Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells.","date":"2020","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/32075880","citation_count":53,"is_preprint":false},{"pmid":"31040242","id":"PMC_31040242","title":"Toxoplasma Controls Host Cyclin E Expression through the Use of a Novel MYR1-Dependent Effector Protein, HCE1.","date":"2019","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/31040242","citation_count":51,"is_preprint":false},{"pmid":"31366709","id":"PMC_31366709","title":"Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase.","date":"2019","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/31366709","citation_count":50,"is_preprint":false},{"pmid":"29615509","id":"PMC_29615509","title":"MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.","date":"2018","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/29615509","citation_count":44,"is_preprint":false},{"pmid":"36309013","id":"PMC_36309013","title":"The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages.","date":"2022","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/36309013","citation_count":42,"is_preprint":false},{"pmid":"32518180","id":"PMC_32518180","title":"Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii.","date":"2020","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/32518180","citation_count":28,"is_preprint":false},{"pmid":"32156805","id":"PMC_32156805","title":"In Vitro Characterization of Protein Effector Export in the Bradyzoite Stage of Toxoplasma gondii.","date":"2020","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/32156805","citation_count":28,"is_preprint":false},{"pmid":"30177926","id":"PMC_30177926","title":"Inhibition of Nitric Oxide Production in Activated Macrophages Caused by Toxoplasma gondii Infection Occurs by Distinct Mechanisms in Different Mouse Macrophage Cell Lines.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30177926","citation_count":26,"is_preprint":false},{"pmid":"34921775","id":"PMC_34921775","title":"Transcriptional modification of host cells harboring Toxoplasma gondii bradyzoites prevents IFN gamma-mediated cell death.","date":"2021","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/34921775","citation_count":23,"is_preprint":false},{"pmid":"32075881","id":"PMC_32075881","title":"The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells.","date":"2020","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/32075881","citation_count":23,"is_preprint":false},{"pmid":"31024505","id":"PMC_31024505","title":"The Virulence-Related MYR1 Protein of Toxoplasma gondii as a Novel DNA Vaccine Against Toxoplasmosis in Mice.","date":"2019","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/31024505","citation_count":21,"is_preprint":false},{"pmid":"33370417","id":"PMC_33370417","title":"Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/33370417","citation_count":20,"is_preprint":false},{"pmid":"32853276","id":"PMC_32853276","title":"Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/32853276","citation_count":16,"is_preprint":false},{"pmid":"27580381","id":"PMC_27580381","title":"Analysis of structures and epitopes of a novel secreted protein MYR1 in Toxoplasma gondii.","date":"2016","source":"Folia parasitologica","url":"https://pubmed.ncbi.nlm.nih.gov/27580381","citation_count":9,"is_preprint":false},{"pmid":"36210525","id":"PMC_36210525","title":"Importance of aspartyl protease 5 in the establishment of the intracellular niche during acute and chronic infection of Toxoplasma gondii.","date":"2022","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/36210525","citation_count":6,"is_preprint":false},{"pmid":"39681694","id":"PMC_39681694","title":"Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism.","date":"2024","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/39681694","citation_count":2,"is_preprint":false},{"pmid":"39654402","id":"PMC_39654402","title":"Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/39654402","citation_count":1,"is_preprint":false},{"pmid":"30279290","id":"PMC_30279290","title":"Erratum for Franco et al., \"A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells\".","date":"2018","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/30279290","citation_count":1,"is_preprint":false},{"pmid":"41959509","id":"PMC_41959509","title":"A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41959509","citation_count":0,"is_preprint":false},{"pmid":"31366706","id":"PMC_31366706","title":"Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host Cell.","date":"2019","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/31366706","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14413,"output_tokens":5111,"usd":0.059952,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11703,"output_tokens":5853,"usd":0.10242,"stage2_stop_reason":"end_turn"},"total_usd":0.162372,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"MYR1 (TGGT1_254470) is a secreted protein that is cleaved by TgASP5 into two stable fragments, both of which localize within the parasitophorous vacuole (PV) and at the parasitophorous vacuole membrane (PVM). Deletion of MYR1 abolishes GRA24 transit to the host nucleus, establishing MYR1 as essential machinery for translocation of dense granule effectors (including GRA16 and GRA24) from the PV into the host cell cytosol and nucleus. MYR1 is also required for host c-Myc upregulation.\",\n      \"method\": \"Whole-genome sequencing of flow-cytometry-selected mutants; immunofluorescence localization; genetic deletion (ΔMyR1) with host c-Myc and GRA24 nuclear transit as phenotypic readouts; TgASP5-dependent cleavage assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and cell-biological approaches, multiple orthogonal readouts (c-Myc induction, GRA24 nuclear localization, GRA16 function), foundational paper replicated by multiple subsequent studies\",\n      \"pmids\": [\"26838724\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MYR1 forms a protein complex at the PVM with MYR2 and MYR3. MYR2 and MYR3 are secreted into the PV space and co-localize with PVM and MYR1. MYR3, but not MYR2, stably associates with MYR1. The N- and C-terminal fragments of MYR1 are disulfide-linked. All three proteins are membrane-associated (consistent with predicted transmembrane domains). MYR2 and MYR3 are required for translocation of a subset of GRAs (GRA16, GRA24) into the host cell.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence co-localization; genetic screens (c-Myc and GRA export assays in mutants); DHFR domain fusion block-of-function assay supporting a translocon model\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal methods (co-localization, genetic epistasis, domain-fusion block), replicated by subsequent MYR complex studies\",\n      \"pmids\": [\"29357375\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNA-Seq of human foreskin fibroblasts infected with RHΔmyr1 vs. wild-type established that the majority of differentially regulated host genes responding to Toxoplasma infection are MYR1-dependent. A subset of MYR1-dependent responses are ASP5-independent, indicating MYR1 function does not strictly require ASP5 cleavage for export of some effectors. MYR1-dependent effects include upregulation of E2F transcription factors/cell cycle genes and downregulation of interferon signaling.\",\n      \"method\": \"RNA-Seq transcriptomics of infected host cells; genetic deletion of MYR1 and ASP5 compared to wild-type\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined transcriptome readout, single lab, single primary method (RNA-Seq)\",\n      \"pmids\": [\"29615509\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ROP17, a rhoptry serine/threonine protein kinase, is required for MYR1-dependent translocation of dense granule effectors across the PVM. Null or catalytically dead ROP17 mutants are defective in GRA translocation. ROP17 acts from the host cytosolic side of the PVM (trans-rescue by ROP17 from co-infecting parasites rescues translocation), placing it as a component upstream of or parallel to the MYR translocon complex.\",\n      \"method\": \"Whole-genome sequencing of translocation-defective mutants; genetic complementation and trans-rescue experiments; catalytic dead mutant construction; immunofluorescence\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — catalytically dead mutant, trans-rescue epistasis, multiple genetic and cell-biological approaches in single study\",\n      \"pmids\": [\"31366709\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HCE1, a novel dense granule effector, is exported from the PV in a MYR1-dependent manner and localizes to the host nucleus, where it binds E2F3, E2F4, and DP1 transcription factors (shown by immunoprecipitation from infected host cells) and is necessary and sufficient to upregulate cyclin E and E2F target genes.\",\n      \"method\": \"Genetic deletion (ΔHce1); immunoprecipitation from infected host cells; expression in uninfected HFFs and Neospora caninum for sufficiency; immunofluorescence\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined nuclear phenotype, Co-IP identifying binding partners, heterologous expression for sufficiency, multiple orthogonal methods\",\n      \"pmids\": [\"31040242\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Affinity purification of MYR1 identified interacting proteins MYR4 (TGGT1_211460), GRA54 (TGGT1_204340), PPM3C (TGGT1_270320), GRA44, and GRA45 within the PV. Of these, GRA44, GRA45, and MYR4 are each essential for GRA16 translocation and host c-Myc/cyclin E1 upregulation, expanding the translocon complex to at least eight discrete proteins.\",\n      \"method\": \"Affinity purification/Co-immunoprecipitation of MYR1 followed by mass spectrometry; genetic deletion of identified interactors with GRA16 translocation and c-Myc/cyclin E1 as functional readouts\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — AP-MS identification of interactors confirmed by genetic deletion with multiple functional readouts, rigorous mechanistic follow-up\",\n      \"pmids\": [\"32075880\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA44, which contains a putative acid phosphatase domain, physically interacts with MYR1 (shown by immunoprecipitation) and is required for host cell c-Myc upregulation downstream of effector secretion, phenocopying loss of the MYR translocon.\",\n      \"method\": \"Immunoprecipitation; conditional knockdown of GRA44; c-Myc upregulation assay\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus conditional KD with functional readout, single lab, moderate replication (consistent with PMID:32075880)\",\n      \"pmids\": [\"32075881\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MYR1 is identified as a component of the cyst wall proteome in vitro. During tachyzoite-to-bradyzoite transition, the export of effectors (GRA16, GRA24, TgIST, and others) into the host nucleus occurs primarily during early time points post-infection, with diminishing nuclear accumulation at later stages. TgIST-mediated suppression of IFN-γ signaling persists in bradyzoite infection, demonstrating continued functional effector export beyond early stages.\",\n      \"method\": \"Epitope tagging of endogenous effector loci; immunofluorescence assays at multiple time points during bradyzoite differentiation; quantification of host nuclear accumulation\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct imaging with epitope-tagged endogenous loci at multiple time points, single lab, IFN-γ suppression functional readout\",\n      \"pmids\": [\"32156805\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TgPPM3C, a PP2C-class serine/threonine phosphatase that co-immunoprecipitates with MYR1, is required for export of GRA16 and GRA28 but not other MYR1-dependent effectors. Phosphoproteomic analysis shows that loss of PPM3C alters phosphorylation of GRA16, GRA28, and MYR1 itself. Phosphomimetic mutation of two GRA16 serine residues causes export defects, indicating that de-phosphorylation by PPM3C is a prerequisite for GRA16 export through the MYR translocon.\",\n      \"method\": \"Genetic deletion of PPM3C; phosphoproteomics; phosphomimetic mutagenesis of GRA16; immunofluorescence for effector nuclear localization; prior Co-IP establishing MYR1 interaction (PMID:32075880)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — combines phosphoproteomic analysis, phosphomimetic mutagenesis, and genetic KO with defined export phenotype; multiple orthogonal methods in single study\",\n      \"pmids\": [\"33370417\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Single-cell transcriptomic analysis of uninfected-injected (U-I) cells versus infected cells from wild-type and ΔMyR1 parasites established that the host's earliest transcriptional response to infection is driven primarily by injected ROPs, not MYR1-dependent GRAs. MYR1-dependent GRA effectors counter-act ROP-driven pro-inflammatory signatures, and MYR1-dependent paracrine effects further modulate inflammatory responses in bystander cells.\",\n      \"method\": \"Single-cell RNA-Seq of flow-sorted U-I, infected, and uninfected cells; genetic deletion of MYR1\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — scRNA-Seq with ΔMyR1 comparison, novel U-I cell enrichment method, single lab\",\n      \"pmids\": [\"32518180\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In bradyzoites, MYR1-dependent protein export is responsible for transcriptional remodeling of host cells, including suppression of type I IFN and IFN-γ signaling pathways. Loss of MYR1 abrogates this transcriptional remodeling and removes protection of host cells from IFN-γ-mediated cell death.\",\n      \"method\": \"Genetic deletion of MYR1 in bradyzoite stage; RNA-Seq; IFN-γ-mediated cell death assays\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with RNA-Seq and functional cell death readout, demonstrates MYR1-dependent export is active and functionally essential in bradyzoite stage\",\n      \"pmids\": [\"34921775\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GRA28, a dense granule effector, is released from the PV via the MYR1 secretory pathway, traffics to the host cell nucleus, and cooperates with host chromatin remodelers to drive CCR7 expression and chemotactic migration of parasitized macrophages. In vivo, MYR1-deficient parasites are outcompeted in dissemination by wild-type parasites.\",\n      \"method\": \"Genetic deletion of MYR1 and GRA28; adoptive transfer in vivo migration assays; immunofluorescence; chromatin remodeler interaction studies\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic deletion of MYR1 and GRA28 with in vitro and in vivo migration phenotype, mechanistic link to chromatin remodeling, multiple orthogonal methods\",\n      \"pmids\": [\"36309013\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ASP5 is required for proteolytic cleavage of MYR1 and other substrates (GRAs) at an ASPH motif in the Golgi, enabling their proper localization within the PV/PVM and their function in the MYR1/2/3 export translocon. Bradyzoite-stage ASP5 substrates also participate in cyst wall formation.\",\n      \"method\": \"N-terminal amine isotopic labeling of substrates (TAILS) to map cleavage sites; genetic deletion of ASP5; comparison of tachyzoite and bradyzoite N-terminomes\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — TAILS N-terminome proteomics plus genetic KO, single lab, provides direct identification of ASP5 cleavage sites on MYR1 pathway components\",\n      \"pmids\": [\"36210525\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MYR1 is required for T. gondii-induced early S-phase arrest and double-strand DNA breaks in primary human host cells within 15 min of infection. ΔMyR1 tachyzoites fail to induce S-phase arrest and DNA damage foci. This MYR1-dependent DNA damage is ROS-independent and is not mediated by HCE1/cyclin E1 overexpression.\",\n      \"method\": \"Genetic deletion of MYR1 and HCE1; cell cycle analysis; γH2AX foci quantification for DNA damage; ROS inhibitor experiments; time-course immunofluorescence\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple cell-biology readouts (cell cycle, DNA damage markers), ROS exclusion controls, single lab\",\n      \"pmids\": [\"39681694\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MYR1-dependent secreted proteins generate a permissive host niche in a paracrine manner: wild-type parasites co-infecting the same host can rescue ΔMYR1 parasite growth in vivo, independent of adaptive immunity or key pro-inflammatory cytokines. MYR1 is not required for parasite survival within IFN-γ-activated macrophages, indicating its primary function is paracrine niche creation rather than protection of the infected cell.\",\n      \"method\": \"Co-infection experiments in mice with WT and ΔMYR1 parasites; immunodeficient mouse models; cytokine knockout mouse models; in vitro macrophage survival assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple in vivo and in vitro genetic experiments with multiple host immune pathway knockouts, rigorous trans-rescue epistasis, replicates prior CRISPR screen observation (PMID:31481656)\",\n      \"pmids\": [\"39654402\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Activation of the non-canonical NF-κB pathway (nuclear accumulation of RelB and p52) during T. gondii infection is strictly MYR1-dependent. Mechanistically, MYR1-dependent effectors drive depletion of the negative regulator TRAF3, leading to NIK stabilization, p100 phosphorylation, and p52 processing. Eight MYR1-dependent effectors (IST, NSM, HCE1/TEEGR, GRA16, GRA18, GRA24, GRA28, GRA84) function additively through this pathway; no single effector is solely responsible.\",\n      \"method\": \"Genetic deletion of MYR1; combinatorial knockout parasites lacking multiple effectors; immunoblot for TRAF3/NIK/p100/p52; immunofluorescence for RelB/p52 nuclear accumulation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with mechanistic downstream readouts, combinatorial KO panel, preprint not yet peer-reviewed\",\n      \"pmids\": [\"41959509\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    }\n  ],\n  \"current_model\": \"MYR1 is a secreted, TgASP5-cleaved protein that localizes to the parasitophorous vacuole membrane (PVM) where it forms the core of a multi-protein translocon complex (with MYR2, MYR3, MYR4, GRA44, GRA45, ROP17, and PPM3C, among others) that is essential for translocation of dense granule effector proteins from the PV lumen across the PVM into the host cell cytosol and nucleus, thereby enabling Toxoplasma to reprogram host transcription (c-Myc, cyclin E, E2F targets, IFN signaling, non-canonical NF-κB), induce early host DNA damage and cell-cycle arrest, drive macrophage chemotactic dissemination, and create a permissive paracrine niche in vivo — all of which are required for full parasite virulence.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MYR1 is a secreted Toxoplasma gondii protein that forms the core of a parasitophorous vacuole membrane (PVM) translocon required for exporting dense granule (GRA) effector proteins from the vacuolar lumen into the host cell cytosol and nucleus, thereby enabling the parasite to reprogram host transcription [#0, #1]. MYR1 is processed by the Golgi aspartyl protease TgASP5 into two disulfide-linked, stably associated fragments that localize to the PV and PVM [#0, #12]. At the PVM it assembles with MYR2, MYR3, and MYR4 and additional interactors GRA44, GRA45, GRA54, and the PP2C phosphatase PPM3C into a multi-protein complex, with MYR3, MYR4, GRA44, and GRA45 each individually required for effector translocation and downstream host c-Myc/cyclin E1 induction [#1, #5, #6]. Translocation additionally depends on the rhoptry kinase ROP17 acting from the host-cytosolic face of the PVM, and on PPM3C-mediated dephosphorylation of specific GRA16 serines as a prerequisite for that effector's export [#3, #8]. Through this pathway MYR1 delivers effectors including GRA16, GRA24, HCE1/TEEGR, GRA28, and TgIST that collectively drive the majority of the host transcriptional response to infection — upregulating E2F/cell-cycle genes, suppressing type I and type II interferon signaling, activating the non-canonical NF-\\u03baB pathway via TRAF3 depletion, inducing early S-phase arrest and DNA double-strand breaks, and driving CCR7-dependent chemotactic dissemination of parasitized macrophages [#2, #4, #10, #11, #13, #15]. In vivo, MYR1-dependent secretion creates a permissive paracrine host niche that supports parasite growth independent of adaptive immunity, establishing this export machinery as central to virulence [#14]. MYR1-dependent effector export persists and remains functionally essential in the bradyzoite stage [#10].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established that Toxoplasma requires a dedicated machinery to move dense granule effectors across the PVM into the host, answering how vacuole-confined parasites reach the host nucleus.\",\n      \"evidence\": \"Whole-genome sequencing of translocation-defective mutants, genetic deletion of MYR1, and TgASP5-dependent cleavage assays with GRA24 nuclear transit and host c-Myc as readouts in RH strain\",\n      \"pmids\": [\"26838724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether MYR1 is a channel, chaperone, or scaffold\", \"Additional complex components unknown\", \"Molecular mechanism of substrate recognition not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined MYR1 as part of a membrane-associated PVM complex, showing the translocon is multi-subunit rather than a single protein.\",\n      \"evidence\": \"Co-immunoprecipitation, immunofluorescence co-localization, genetic epistasis, and DHFR domain-fusion block-of-function assays identifying MYR2/MYR3 in RH strain\",\n      \"pmids\": [\"29357375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the complex unresolved\", \"MYR2 association mode with MYR1 unclear (does not stably bind)\", \"Mechanism of substrate passage not shown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Quantified the scope of MYR1 control over the host response, showing most infection-induced host gene changes depend on MYR1 and that some effects are ASP5-independent.\",\n      \"evidence\": \"RNA-Seq of infected human fibroblasts comparing wild-type, \\u0394myr1, and \\u0394asp5 in RH strain\",\n      \"pmids\": [\"29615509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Individual effectors responsible for each transcriptional signature not mapped\", \"Mechanism of ASP5-independent export undefined\", \"Single-method (RNA-Seq) study\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified a kinase requirement acting from the host side of the PVM, revealing translocation depends on phosphoregulation across the membrane.\",\n      \"evidence\": \"Whole-genome sequencing of mutants, catalytically-dead ROP17 mutants, and trans-rescue complementation in RH strain\",\n      \"pmids\": [\"31366709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ROP17 substrates in the translocation process not identified\", \"Whether ROP17 acts on the translocon or on effectors unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked a specific MYR1-exported effector to host cell-cycle reprogramming, giving mechanistic identity to the E2F/cyclin E transcriptional signature.\",\n      \"evidence\": \"Genetic deletion of HCE1, Co-IP identifying E2F3/E2F4/DP1 binding, and heterologous expression for sufficiency in RH strain\",\n      \"pmids\": [\"31040242\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of HCE1–E2F/DP1 interaction unknown\", \"Does not explain the DNA-damage arm of the response\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Expanded the translocon to at least eight subunits by unbiased purification, defining the full complex needed for export.\",\n      \"evidence\": \"Affinity purification/Co-IP of MYR1 with mass spectrometry plus genetic deletion of MYR4/GRA44/GRA45 with GRA16 translocation and c-Myc/cyclin E1 readouts in RH strain\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Roles of GRA54 not functionally defined\", \"Enzymatic activity of GRA44 acid phosphatase domain not tested directly\", \"Complex assembly order unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed that dephosphorylation is a gating step for export, revealing PPM3C-mediated GRA16 dephosphorylation is a prerequisite for translocation of a subset of effectors.\",\n      \"evidence\": \"Genetic deletion of PPM3C, phosphoproteomics, and phosphomimetic mutagenesis of GRA16 serines with nuclear-localization readouts\",\n      \"pmids\": [\"33370417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why only a subset of effectors (GRA16, GRA28) require PPM3C is unclear\", \"Kinase counteracting PPM3C not identified\", \"Functional consequence of MYR1 phosphorylation change not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the temporal hierarchy of host manipulation, showing injected ROPs drive the earliest response while MYR1-dependent GRAs counteract ROP-driven inflammation, including in bystander cells.\",\n      \"evidence\": \"Single-cell RNA-Seq of flow-sorted uninfected-injected, infected, and uninfected cells with \\u0394myr1 comparison in RH strain\",\n      \"pmids\": [\"32518180\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effectors mediating paracrine effects not identified\", \"Single-lab scRNA-Seq\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed MYR1 in the ASP5 substrate map and cyst wall proteome, and showed effector export operates through bradyzoite differentiation.\",\n      \"evidence\": \"TAILS N-terminomics with ASP5 KO, cyst wall proteomics, and time-course imaging of epitope-tagged effectors during bradyzoite transition\",\n      \"pmids\": [\"32156805\", \"36210525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of MYR1 in the cyst wall vs. translocation not separated\", \"Kinetics of complex turnover across stages unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated MYR1-dependent export is functionally essential in the persistent bradyzoite stage, protecting host cells from IFN-\\u03b3-mediated death.\",\n      \"evidence\": \"Genetic deletion of MYR1 in bradyzoites, RNA-Seq, and IFN-\\u03b3-mediated cell death assays\",\n      \"pmids\": [\"34921775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Individual effectors driving bradyzoite-stage protection not fully mapped\", \"In vivo relevance during chronic infection not addressed here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected MYR1-dependent effector export to organismal dissemination, showing GRA28 drives CCR7-dependent macrophage migration and MYR1 loss impairs in vivo spread.\",\n      \"evidence\": \"Genetic deletion of MYR1 and GRA28, chromatin-remodeler interaction studies, and adoptive-transfer in vivo migration assays\",\n      \"pmids\": [\"36309013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin remodeler identities and mechanism at the CCR7 locus not fully defined\", \"Contribution of migration vs. other MYR1 functions to dissemination not dissected\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Uncovered a rapid MYR1-dependent host DNA-damage and cell-cycle arrest program independent of the HCE1/cyclin E axis, showing MYR1 controls damage responses beyond transcriptional reprogramming.\",\n      \"evidence\": \"Genetic deletion of MYR1 and HCE1, \\u03b3H2AX foci quantification, cell-cycle analysis, and ROS-inhibitor controls in RH strain\",\n      \"pmids\": [\"39681694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effector responsible for DNA damage not identified\", \"Molecular trigger of double-strand breaks unknown\", \"Single-lab study\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the ultimate in vivo purpose of MYR1 secretion as paracrine niche creation rather than protection of the infected cell.\",\n      \"evidence\": \"WT/\\u0394MYR1 co-infection trans-rescue in mice, immunodeficient and cytokine-knockout models, and in vitro macrophage survival assays in RH strain\",\n      \"pmids\": [\"39654402\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Secreted factors mediating the paracrine niche not identified\", \"Host receptors/pathways receiving the paracrine signal unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Assigned MYR1-dependent effectors to non-canonical NF-\\u03baB activation, showing eight effectors act additively to deplete TRAF3 and process p52.\",\n      \"evidence\": \"Genetic deletion of MYR1, combinatorial multi-effector knockout parasites, and immunoblot/immunofluorescence for TRAF3/NIK/p100/p52 and RelB/p52 nuclear accumulation in RH and ME49 (preprint)\",\n      \"pmids\": [\"41959509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Direct molecular mechanism by which each effector depletes TRAF3 not resolved\", \"Relative contribution of individual effectors not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The biophysical mechanism of substrate passage across the PVM — how the MYR translocon recognizes, unfolds, and threads effectors — remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the translocon\", \"Channel vs. chaperone mechanism unresolved\", \"Energetics and driving force of translocation unknown\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0043495\", \"supporting_discovery_ids\": [0, 1, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0065002\", \"supporting_discovery_ids\": [0, 1, 3, 5, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0, 1, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140321\", \"supporting_discovery_ids\": [0, 2, 9, 10, 15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0009405\", \"supporting_discovery_ids\": [11, 14], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [\n      \"MYR translocon complex at the PVM: MYR1, MYR2, MYR3, MYR4, GRA44, GRA45, GRA54, PPM3C [#1, #5]\"\n    ],\n    \"partners\": [\n      \"MYR3 (stable association with MYR1) [#1]\",\n      \"MYR2 [#1]\",\n      \"MYR4 [#5]\",\n      \"GRA44 (putative acid phosphatase domain) [#5, #6]\",\n      \"GRA45 [#5]\",\n      \"GRA54 [#5]\",\n      \"PPM3C (PP2C-class phosphatase) [#5, #8]\",\n      \"ROP17 (rhoptry kinase, acts from host-cytosolic face) [#3]\",\n      \"TgASP5 (protease that cleaves MYR1) [#0, #12]\"\n    ],\n    \"other_free_text\": [\n      \"MYR1 is cleaved by TgASP5 into two disulfide-linked fragments that both localize to the PV/PVM [#0, #1, #12].\",\n      \"MYR1-dependent exported effectors include GRA16, GRA24, HCE1/TEEGR, GRA28, TgIST, GRA18, NSM, GRA84 [#0, #4, #8, #11, #15].\",\n      \"PPM3C-mediated dephosphorylation of specific GRA16 serines is a prerequisite for export of a subset of effectors [#8].\",\n      \"MYR1-dependent host outcomes: c-Myc/cyclin E induction, E2F target upregulation, interferon suppression, non-canonical NF-\\u03baB activation via TRAF3 depletion, early S-phase arrest and DNA double-strand breaks, CCR7-driven macrophage chemotaxis, and paracrine niche creation [#0, #2, #4, #10, #11, #13, #14, #15].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_254470","name":"MYR1","product":"Myc-regulation protein MYR1","uniprot_acc":"S8GT06","chromosome":"TGME49_chrIII","start_pos":1758400},{"strain":"GT1","gene_id":"TGGT1_254470","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrIII","start_pos":1739403},{"strain":"RH88","gene_id":"TGRH88_003880","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023084","start_pos":843996},{"strain":"VEG","gene_id":"TGVEG_254470","name":null,"product":"putative transmembrane protein","uniprot_acc":"V5B3J8","chromosome":"TGVEG_chrIII","start_pos":1767450}]}