{"gene":"TG_319560","gene_symbol":"MIC3","run_date":"2026-09-08T18:10:00+00:00","timeline":{"discoveries":[{"year":2000,"finding":"MIC3 is a secretory adhesin that binds to the surface of host cells and is relocalized to the parasite surface during invasion; the protein contains five partially overlapping EGF-like domains and a chitin binding-like (CBL) domain; it is a homodimer of two ~38-kDa polypeptides linked by disulfide bridges, processed from a 40-kDa precursor.","method":"Western blot adhesion assay with antibody blockade, affinity purification, N-terminal sequencing, immunofluorescence of invading parasites, cDNA cloning and sequence analysis","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding assay, antibody blockade, affinity purification, IFA, sequencing) in a single rigorous study; replicated by independent earlier biochemical characterization (PMID:1944419)","pmids":["11207591","1944419"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"MIC3 requires pro-peptide cleavage to expose its receptor-binding site in the N-terminal chitin binding-like (CBL) domain, and requires dimerization (mediated by the C-terminal domain) for host cell binding; the dimerization domain can convert MIC8 (normally monomeric) into a dimer capable of interacting with host cell receptors.","method":"Transfection of mammalian cells with complete or truncated MIC3 gene sequences, MIC3-binding assay, site-directed mutagenesis of processing and dimerization domains","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution-type cell-based binding assay combined with deletion/truncation mutagenesis and domain-swap experiment; multiple orthogonal approaches in one rigorous study","pmids":["12032066"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"TgMIC8 acts as a transmembrane escorter for the soluble adhesin TgMIC3, sorting it to the micronemes via signals in MIC8's cytoplasmic tail; MIC8 is proteolytically cleaved near its C-terminal domain upon microneme discharge.","method":"Co-localization, functional complementation, topology analysis, processing assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — established escorter function by co-localization and functional evidence within a single lab; single paper but multiple methods","pmids":["11861763"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"GFP fusions to MIC3 are correctly targeted to micronemes in living parasites; deletions within the cysteine-rich central region cause MIC3 to be arrested at various locations in the secretory pathway, suggesting this region is required for proper folding and microneme targeting; misfolded/mistargeted MIC3 is rerouted to the dense granule default secretory pathway.","method":"GFP fusion protein expression, fluorescence microscopy, deletion analysis in transfected parasites","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell imaging and deletion analysis; single lab but multiple constructs tested","pmids":["11254953"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"PRP1 (a parafusin orthologue) is dephosphorylated upon Ca2+-stimulated microneme exocytosis, concurrent with translocation of MIC3 from the parasite apex; quantitative microscopy during invasion showed that both PRP1 and MIC3 fluorescence intensities decrease ~10-fold immediately after invasion, indicating coordinated microneme secretion accompanies cell entry; MIC3 fluorescence recovers to apical distribution after ~15 min post-invasion.","method":"In vivo 32P labeling, immunoblotting, quantitative immunofluorescence microscopy, ethanol-stimulated exocytosis assay, immunoelectron microscopy","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (metabolic labeling, quantitative microscopy, immunoelectron microscopy) from a single lab","pmids":["12925131"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2005,"finding":"Single amino acid substitutions in the CBL domain of MIC3 that abolish host cell binding attenuate virulence in mice; MIC1 and MIC3 act synergistically in virulence — double knockout (mic1/mic3) causes severe virulence attenuation and provides cross-protection, while single knockouts only slightly reduce virulence; MIC3 deletion alone has no effect on invasion in fibroblasts.","method":"Gene disruption (single and double KO), site-directed mutagenesis of CBL domain, in vitro invasion assay, mouse virulence assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockouts combined with mutagenesis and multiple functional readouts (invasion assay, virulence, protection); multiple orthogonal approaches","pmids":["15684324"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2008,"finding":"MIC3 propeptide cleavage occurs in a post-Golgi compartment; the propeptide and EGF modules together encode microneme-targeting information — the minimal targeting signal requires the propeptide plus any one of the three EGF domains; pro-peptide cleavage, dimerization, and the CBL domain (required for host cell binding and virulence) are dispensable for microneme targeting; a fraction of MIC3 is retained in the secretory pathway in a cell-cycle-dependent manner.","method":"Deletion mutant analysis, chimeric protein transfection, pulse-chase and processing assays, fluorescence microscopy","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic deletion and domain-swap mutagenesis with multiple constructs and orthogonal readouts in a single rigorous study","pmids":["18390648"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgROM4 (a rhomboid protease) is required for shedding of MIC3 from the parasite surface; conditional knockdown of ROM4 increased surface expression of MIC3 and reduced its release, disrupted normal gliding motility, and severely impaired invasion despite enhanced host cell binding.","method":"Conditional knockout of ROM4, surface adhesin release assay, flow cytometry, gliding motility assay, invasion assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockdown with multiple orthogonal functional readouts (proteolysis assay, motility, invasion); single lab but rigorous controls","pmids":["20421941"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2011,"finding":"Propeptide domains are functionally interchangeable between T. gondii MICs and with Eimeria tenella EtMIC5; a conserved aliphatic residue (valine at +3 for proM2AP; leucine at +1 for proMIC5) is critical for microneme targeting; MIC3 propeptide contains a valine or leucine near its N-terminus, and mutagenesis of this residue impairs microneme targeting.","method":"N-terminal deletion and point mutagenesis of propeptides, domain-swap chimeras, fluorescence microscopy in transfected parasites","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional trafficking readout; MIC3 finding is one component of a broader systematic study","pmids":["21438967"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"finding":"MIC3 (containing EGF domains) promotes EGFR-Akt activation in host cells; recombinant MIC3 activates EGFR-Akt signaling; parasites lacking MIC3 (mic3 ko) show impaired EGFR-Akt activation; EGFR activation prevents autophagic targeting of the parasitophorous vacuole, and recombinant MIC3 can inhibit parasite killing triggered by CD154 in autophagy-stimulated cells.","method":"Knockout parasite infection, recombinant protein treatment, EGFR/Akt phosphorylation assays, LC3 accumulation assay, parasite killing assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout combined with recombinant protein rescue, multiple cell types, multiple signaling readouts and functional consequence (parasite killing)","pmids":["24367261"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"MIC3 binds to host proteins Spata3 (spermatogenesis-associated protein 3) and Dkk2 (dickkopf-related protein 2); the tandem repeat EGF domains of MIC3 are critical for mediating these interactions.","method":"Yeast two-hybrid screen using MIC3 as bait against mouse protein library, confirmed by additional protein-protein interaction assays","journal":"Parasitology research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus confirmation assay; interactions validated by secondary tests but no functional consequence in parasite demonstrated","pmids":["27053129"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"MIC3 is upregulated ~5-fold in the highly virulent RH strain compared with the less virulent TgCtwh3 (Chinese 1) strain; recombinant MIC3 experimentally induces TNF-α secretory response and macrophage M1 polarization.","method":"iTRAQ quantitative proteomics of excretory-secretory antigens, recombinant protein stimulation of macrophages, TNF-α ELISA, M1 polarization assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative proteomics combined with recombinant protein functional assay; single lab, two orthogonal approaches","pmids":["28000706"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH; TgCtwh3"}]},{"year":2019,"finding":"MIC3 binds heparan sulfate (heparin); recombinant TgMIC3 shows specific binding to heparin and host cell surfaces; pre-incubation of host cells with recombinant MIC3 blocks T. gondii invasion; MIC3 expression is higher in invading parasites compared to free-ranging parasites.","method":"Heparin-binding assay, host cell surface binding assay with recombinant protein, competitive invasion inhibition assay, antibody-based invasion blockade, RT-PCR/Western blot expression analysis","journal":"Acta tropica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding assays with functional invasion inhibition readout; single lab","pmids":["30664845"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2023,"finding":"A MIC3 peptide induces TNF-α production, NF-κB phosphorylation, iNOS transcription, and Ly6C expression in mouse macrophages via the TLR11/MyD88/NF-κB pathway; MIC3 can adhere to the macrophage cell membrane and enter cells; MIC3 does not induce these responses in human THP-1 macrophages (which lack TLR11); MIC3 does not induce IL-12p40 (unlike the canonical TLR11 ligand profilin), suggesting MIC3 is a distinct TLR11 ligand.","method":"MyD88 inhibitor treatment, siRNA knockdown of Tlr11, CRISPR/Cas9 Tlr11 knockout in RAW264.7 cells, NF-κB phosphorylation assay, ELISA for TNF-α, qRT-PCR for iNOS and Ly6C, immunofluorescence for MIC3 localization in macrophages","journal":"PLoS neglected tropical diseases","confidence":"High","confidence_rationale":"Tier 2 / Strong — three independent genetic/pharmacological methods to disrupt TLR11/MyD88 pathway (inhibitor, siRNA, CRISPR KO) with consistent results; multiple signaling readouts; cross-species (mouse vs. human) validation","pmids":["36730424"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgMIC3 is a micronemal homodimeric adhesin that is synthesized with a pro-peptide (cleaved in a post-Golgi compartment), targeted to micronemes via signals in both its pro-peptide and EGF domains, secreted onto the parasite surface during invasion where its N-terminal chitin binding-like (CBL) domain mediates host cell receptor binding (dependent on prior pro-peptide cleavage and dimerization), shed from the surface by the rhomboid protease TgROM4, and—once secreted into the host cell environment—activates EGFR-Akt signaling (via its EGF domains) to suppress autophagic killing of the parasite, and also engages host TLR11/MyD88/NF-κB signaling to induce TNF-α and macrophage M1 polarization; MIC3 acts synergistically with MIC1 to promote virulence, and its transmembrane escorter is TgMIC8."},"narrative":{"mechanistic_narrative":"TgMIC3 is a micronemal secretory adhesin of Toxoplasma gondii that couples host-cell attachment during invasion to modulation of host innate immunity [PMID:11207591, PMID:1944419, PMID:24367261]. It is synthesized as a 40-kDa precursor and matures into a disulfide-linked homodimer of ~38-kDa polypeptides bearing five partially overlapping EGF-like domains and an N-terminal chitin binding-like (CBL) domain, and it is relocalized from the parasite apex onto the surface during cell entry [PMID:11207591, PMID:1944419, PMID:12925131]. Receptor binding requires two prior maturation events: cleavage of the pro-peptide to expose the CBL domain, and C-terminal-domain-mediated dimerization; the dimerization module is potent enough to convert the normally monomeric MIC8 into a receptor-competent dimer [PMID:12032066]. The CBL domain engages host cell receptors, including heparan sulfate, and single substitutions that abolish CBL binding attenuate virulence, establishing adhesion as the effector activity relevant to pathogenesis [PMID:15684324, PMID:30664845]. Microneme targeting is encoded jointly by the pro-peptide and the EGF modules—the minimal signal being the pro-peptide plus any one EGF domain—and depends on a conserved aliphatic residue near the pro-peptide N-terminus, whereas pro-peptide cleavage, dimerization, and the CBL domain are dispensable for trafficking; the soluble adhesin reaches the micronemes as cargo of the transmembrane escorter MIC8, and is shed from the parasite surface by the rhomboid protease TgROM4 to permit productive invasion [PMID:18390648, PMID:21438967, PMID:11861763, PMID:20421941]. Beyond adhesion, secreted MIC3 acts on the host: its EGF domains activate EGFR-Akt signaling to suppress autophagic killing of the parasite, and a MIC3 peptide drives TNF-α production, NF-κB activation, iNOS and Ly6C induction, and M1 macrophage polarization through the TLR11/MyD88/NF-κB pathway [PMID:24367261, PMID:28000706, PMID:36730424]. MIC3 acts synergistically with MIC1, and only combined mic1/mic3 disruption severely attenuates virulence [PMID:15684324].","teleology":[{"year":2000,"claim":"Whether MIC3 was a structural micronemal protein or a functional adhesin was unresolved; biochemical and imaging characterization established it as a disulfide-linked homodimeric adhesin that binds host cells and redistributes to the parasite surface during invasion.","evidence":"adhesion assays with antibody blockade, affinity purification, N-terminal sequencing, invasion IFA, and cDNA cloning in T. gondii","pmids":["11207591","1944419"],"confidence":"High","gaps":["Host receptor identity not defined","Contribution of individual domains to binding not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"claim":"It was unknown what directed MIC3 to the micronemes; live-cell GFP-fusion and deletion analysis showed the cysteine-rich central region is required for proper folding and micronemal targeting, with misfolded protein defaulting to the dense-granule pathway.","evidence":"GFP-fusion imaging and deletion analysis in transfected T. gondii","pmids":["11254953"],"confidence":"Medium","gaps":["Minimal targeting determinant not yet defined","Molecular recognition of the signal not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"How a soluble adhesin with no membrane anchor reaches the micronemes was unclear; MIC8 was shown to act as a transmembrane escorter that sorts MIC3 via cytoplasmic-tail signals and is cleaved upon discharge.","evidence":"co-localization, functional complementation, and processing assays in T. gondii","pmids":["11861763"],"confidence":"Medium","gaps":["Direct MIC3-MIC8 binding interface not mapped","Stoichiometry of the escort complex unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"The requirements for MIC3 to acquire binding activity were unknown; cell-based reconstitution with truncation and domain-swap mutagenesis showed pro-peptide cleavage exposes the CBL receptor-binding site and C-terminal-domain dimerization is required for host-cell binding.","evidence":"mammalian-cell expression, MIC3-binding assays, and site-directed/domain-swap mutagenesis","pmids":["12032066"],"confidence":"High","gaps":["Host receptor engaged by the CBL domain not identified here","Structural basis of dimer-dependent binding not solved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Whether MIC3 relocalization was coordinated with regulated microneme exocytosis was untested; quantitative imaging tied MIC3 apical loss to Ca2+-stimulated secretion accompanying invasion, with recovery ~15 min later.","evidence":"in vivo 32P labeling, quantitative IFA, stimulated-exocytosis and immunoelectron microscopy in T. gondii","pmids":["12925131"],"confidence":"Medium","gaps":["Molecular trigger linking Ca2+ signaling to MIC3 discharge not defined","Fate of surface MIC3 during recovery unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2005,"claim":"Whether MIC3 binding activity is functionally important in vivo was unknown; CBL point mutants and single/double knockouts established that CBL-mediated binding contributes to virulence and that MIC1 and MIC3 act synergistically, though MIC3 is dispensable for fibroblast invasion.","evidence":"single/double gene disruption, CBL-domain mutagenesis, invasion assays, and mouse virulence assays in T. gondii RH","pmids":["15684324"],"confidence":"High","gaps":["Molecular basis of MIC1/MIC3 synergy not defined","Reason for dispensability in fibroblast invasion unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2008,"claim":"The precise trafficking determinants and processing compartment were unresolved; systematic deletion and chimera analysis localized pro-peptide cleavage to a post-Golgi compartment and defined the minimal micronemal signal as pro-peptide plus any one EGF domain, distinct from the domains needed for binding.","evidence":"deletion mutants, chimeric proteins, and pulse-chase processing assays in T. gondii","pmids":["18390648"],"confidence":"High","gaps":["Identity of the pro-peptide-cleaving protease not established","Basis of cell-cycle-dependent secretory retention unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"How MIC3 is removed from the surface to allow invasion was unknown; conditional ROM4 knockdown showed the rhomboid protease TgROM4 sheds MIC3, and its loss increases surface MIC3, impairs gliding and invasion despite enhanced binding.","evidence":"conditional ROM4 knockdown with adhesin-release, flow cytometry, motility and invasion assays in T. gondii RH","pmids":["20421941"],"confidence":"High","gaps":["MIC3 cleavage site for ROM4 not mapped","Whether MIC3 is a direct vs. indirect ROM4 substrate not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2011,"claim":"It was unknown whether the pro-peptide targeting determinant was conserved across coccidia; mutagenesis showed pro-peptides are interchangeable across MICs and with Eimeria EtMIC5 and that a conserved aliphatic residue near the N-terminus is critical for MIC3 microneme targeting.","evidence":"N-terminal deletion/point mutagenesis and domain-swap chimeras in transfected T. gondii","pmids":["21438967"],"confidence":"Medium","gaps":["Receptor recognizing the aliphatic-residue signal not identified","MIC3-specific data are one component of a broader study"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"claim":"Whether MIC3 has host-signaling functions beyond adhesion was unknown; knockout and recombinant-protein experiments showed its EGF domains activate EGFR-Akt signaling that blocks autophagic targeting and killing of the parasite.","evidence":"mic3 KO infection, recombinant MIC3 treatment, EGFR/Akt phosphorylation, LC3 accumulation and parasite-killing assays","pmids":["24367261"],"confidence":"High","gaps":["Direct MIC3-EGFR contact not demonstrated","In vivo relevance of the EGFR-autophagy axis not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Host binding partners of the EGF domains beyond EGFR were unknown; a yeast two-hybrid screen identified Spata3 and Dkk2 as MIC3 interactors mediated by the tandem EGF repeats.","evidence":"yeast two-hybrid screen against a mouse library with confirmatory interaction assays","pmids":["27053129"],"confidence":"Medium","gaps":["No functional consequence of Spata3/Dkk2 binding demonstrated in the parasite","Interactions not validated in an infection context"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Whether MIC3 abundance and immunostimulatory activity relate to strain virulence was untested; iTRAQ proteomics found MIC3 ~5-fold higher in virulent RH than in TgCtwh3, and recombinant MIC3 induced TNF-α and M1 macrophage polarization.","evidence":"iTRAQ proteomics of secreted antigens and recombinant-protein macrophage stimulation with TNF-α ELISA and M1 assays","pmids":["28000706"],"confidence":"Medium","gaps":["Receptor/pathway driving TNF-α and M1 polarization not defined here","Causal link between MIC3 abundance and strain virulence not proven"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH; TgCtwh3"}]},{"year":2019,"claim":"The identity of a host receptor for the CBL/adhesin activity was still open; recombinant MIC3 was shown to bind heparan sulfate and host cell surfaces, and to competitively block invasion, with higher expression in invading parasites.","evidence":"heparin- and cell-surface binding assays, competitive and antibody-mediated invasion inhibition, and expression analysis in T. gondii RH","pmids":["30664845"],"confidence":"Medium","gaps":["Whether heparan sulfate is the physiological invasion receptor not established","Domain responsible for heparin binding not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2023,"claim":"The receptor and pathway for MIC3's pro-inflammatory activity were undefined; inhibitor, siRNA and CRISPR-KO approaches showed a MIC3 peptide drives TNF-α, NF-κB, iNOS and Ly6C via TLR11/MyD88/NF-κB in mouse macrophages but not in TLR11-deficient human cells, and it is a TLR11 ligand distinct from profilin (no IL-12p40 induction).","evidence":"MyD88 inhibition, Tlr11 siRNA and CRISPR KO in RAW264.7, NF-κB/TNF-α/iNOS/Ly6C readouts, and mouse-vs-human comparison","pmids":["36730424"],"confidence":"High","gaps":["Direct MIC3-TLR11 binding not demonstrated","Human host receptor for MIC3 immunostimulation unknown","Role of full-length MIC3 vs. peptide in vivo not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The physiological host receptor engaged by the CBL domain during invasion, the direct structural basis for EGFR and TLR11 engagement, and the mechanism of MIC1/MIC3 virulence synergy remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No solved structure of MIC3 in complex with any host receptor","Direct MIC3-EGFR and MIC3-TLR11 contacts not demonstrated","Molecular basis of MIC1/MIC3 synergy undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MIC3 homodimer (disulfide-linked ~38-kDa polypeptides)","MIC3-MIC8 escort complex"],"partners":["MIC8 (TRANSMEMBRANE ESCORTER)","MIC1 (SYNERGISTIC VIRULENCE PARTNER)","TGROM4 (RHOMBOID PROTEASE THAT SHEDS MIC3)","HOST EGFR","HOST TLR11/MYD88","HOST HEPARAN SULFATE","HOST SPATA3","HOST DKK2"],"other_free_text":["Synthesized as a 40-kDa precursor with a pro-peptide cleaved in a post-Golgi compartment; pro-peptide cleavage exposes the CBL receptor-binding site.","Microneme targeting encoded by pro-peptide plus any one EGF domain and a conserved N-terminal aliphatic residue; pro-peptide cleavage, dimerization and CBL domain are dispensable for targeting.","MIC3 is a TLR11 ligand distinct from profilin (induces TNF-α but not IL-12p40) and is active in mouse but not TLR11-deficient human macrophages.","MIC3 deletion alone does not impair fibroblast invasion, but CBL binding mutations and combined mic1/mic3 loss attenuate virulence."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"11861763","id":"PMC_11861763","title":"A family of transmembrane microneme proteins of Toxoplasma gondii contain EGF-like domains and function as escorters.","date":"2002","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/11861763","citation_count":135,"is_preprint":false},{"pmid":"15684324","id":"PMC_15684324","title":"Synergistic role of micronemal proteins in Toxoplasma gondii virulence.","date":"2005","source":"The 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\"method\": \"Western blot adhesion assay with antibody blockade, affinity purification, N-terminal sequencing, immunofluorescence of invading parasites, cDNA cloning and sequence analysis\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding assay, antibody blockade, affinity purification, IFA, sequencing) in a single rigorous study; replicated by independent earlier biochemical characterization (PMID:1944419)\",\n      \"pmids\": [\"11207591\", \"1944419\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MIC3 requires pro-peptide cleavage to expose its receptor-binding site in the N-terminal chitin binding-like (CBL) domain, and requires dimerization (mediated by the C-terminal domain) for host cell binding; the dimerization domain can convert MIC8 (normally monomeric) into a dimer capable of interacting with host cell receptors.\",\n      \"method\": \"Transfection of mammalian cells with complete or truncated MIC3 gene sequences, MIC3-binding assay, site-directed mutagenesis of processing and dimerization domains\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution-type cell-based binding assay combined with deletion/truncation mutagenesis and domain-swap experiment; multiple orthogonal approaches in one rigorous study\",\n      \"pmids\": [\"12032066\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TgMIC8 acts as a transmembrane escorter for the soluble adhesin TgMIC3, sorting it to the micronemes via signals in MIC8's cytoplasmic tail; MIC8 is proteolytically cleaved near its C-terminal domain upon microneme discharge.\",\n      \"method\": \"Co-localization, functional complementation, topology analysis, processing assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — established escorter function by co-localization and functional evidence within a single lab; single paper but multiple methods\",\n      \"pmids\": [\"11861763\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"GFP fusions to MIC3 are correctly targeted to micronemes in living parasites; deletions within the cysteine-rich central region cause MIC3 to be arrested at various locations in the secretory pathway, suggesting this region is required for proper folding and microneme targeting; misfolded/mistargeted MIC3 is rerouted to the dense granule default secretory pathway.\",\n      \"method\": \"GFP fusion protein expression, fluorescence microscopy, deletion analysis in transfected parasites\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell imaging and deletion analysis; single lab but multiple constructs tested\",\n      \"pmids\": [\"11254953\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PRP1 (a parafusin orthologue) is dephosphorylated upon Ca2+-stimulated microneme exocytosis, concurrent with translocation of MIC3 from the parasite apex; quantitative microscopy during invasion showed that both PRP1 and MIC3 fluorescence intensities decrease ~10-fold immediately after invasion, indicating coordinated microneme secretion accompanies cell entry; MIC3 fluorescence recovers to apical distribution after ~15 min post-invasion.\",\n      \"method\": \"In vivo 32P labeling, immunoblotting, quantitative immunofluorescence microscopy, ethanol-stimulated exocytosis assay, immunoelectron microscopy\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (metabolic labeling, quantitative microscopy, immunoelectron microscopy) from a single lab\",\n      \"pmids\": [\"12925131\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Single amino acid substitutions in the CBL domain of MIC3 that abolish host cell binding attenuate virulence in mice; MIC1 and MIC3 act synergistically in virulence — double knockout (mic1/mic3) causes severe virulence attenuation and provides cross-protection, while single knockouts only slightly reduce virulence; MIC3 deletion alone has no effect on invasion in fibroblasts.\",\n      \"method\": \"Gene disruption (single and double KO), site-directed mutagenesis of CBL domain, in vitro invasion assay, mouse virulence assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockouts combined with mutagenesis and multiple functional readouts (invasion assay, virulence, protection); multiple orthogonal approaches\",\n      \"pmids\": [\"15684324\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MIC3 propeptide cleavage occurs in a post-Golgi compartment; the propeptide and EGF modules together encode microneme-targeting information — the minimal targeting signal requires the propeptide plus any one of the three EGF domains; pro-peptide cleavage, dimerization, and the CBL domain (required for host cell binding and virulence) are dispensable for microneme targeting; a fraction of MIC3 is retained in the secretory pathway in a cell-cycle-dependent manner.\",\n      \"method\": \"Deletion mutant analysis, chimeric protein transfection, pulse-chase and processing assays, fluorescence microscopy\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic deletion and domain-swap mutagenesis with multiple constructs and orthogonal readouts in a single rigorous study\",\n      \"pmids\": [\"18390648\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgROM4 (a rhomboid protease) is required for shedding of MIC3 from the parasite surface; conditional knockdown of ROM4 increased surface expression of MIC3 and reduced its release, disrupted normal gliding motility, and severely impaired invasion despite enhanced host cell binding.\",\n      \"method\": \"Conditional knockout of ROM4, surface adhesin release assay, flow cytometry, gliding motility assay, invasion assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockdown with multiple orthogonal functional readouts (proteolysis assay, motility, invasion); single lab but rigorous controls\",\n      \"pmids\": [\"20421941\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Propeptide domains are functionally interchangeable between T. gondii MICs and with Eimeria tenella EtMIC5; a conserved aliphatic residue (valine at +3 for proM2AP; leucine at +1 for proMIC5) is critical for microneme targeting; MIC3 propeptide contains a valine or leucine near its N-terminus, and mutagenesis of this residue impairs microneme targeting.\",\n      \"method\": \"N-terminal deletion and point mutagenesis of propeptides, domain-swap chimeras, fluorescence microscopy in transfected parasites\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional trafficking readout; MIC3 finding is one component of a broader systematic study\",\n      \"pmids\": [\"21438967\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MIC3 (containing EGF domains) promotes EGFR-Akt activation in host cells; recombinant MIC3 activates EGFR-Akt signaling; parasites lacking MIC3 (mic3 ko) show impaired EGFR-Akt activation; EGFR activation prevents autophagic targeting of the parasitophorous vacuole, and recombinant MIC3 can inhibit parasite killing triggered by CD154 in autophagy-stimulated cells.\",\n      \"method\": \"Knockout parasite infection, recombinant protein treatment, EGFR/Akt phosphorylation assays, LC3 accumulation assay, parasite killing assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout combined with recombinant protein rescue, multiple cell types, multiple signaling readouts and functional consequence (parasite killing)\",\n      \"pmids\": [\"24367261\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MIC3 binds to host proteins Spata3 (spermatogenesis-associated protein 3) and Dkk2 (dickkopf-related protein 2); the tandem repeat EGF domains of MIC3 are critical for mediating these interactions.\",\n      \"method\": \"Yeast two-hybrid screen using MIC3 as bait against mouse protein library, confirmed by additional protein-protein interaction assays\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus confirmation assay; interactions validated by secondary tests but no functional consequence in parasite demonstrated\",\n      \"pmids\": [\"27053129\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MIC3 is upregulated ~5-fold in the highly virulent RH strain compared with the less virulent TgCtwh3 (Chinese 1) strain; recombinant MIC3 experimentally induces TNF-α secretory response and macrophage M1 polarization.\",\n      \"method\": \"iTRAQ quantitative proteomics of excretory-secretory antigens, recombinant protein stimulation of macrophages, TNF-α ELISA, M1 polarization assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative proteomics combined with recombinant protein functional assay; single lab, two orthogonal approaches\",\n      \"pmids\": [\"28000706\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH; TgCtwh3\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MIC3 binds heparan sulfate (heparin); recombinant TgMIC3 shows specific binding to heparin and host cell surfaces; pre-incubation of host cells with recombinant MIC3 blocks T. gondii invasion; MIC3 expression is higher in invading parasites compared to free-ranging parasites.\",\n      \"method\": \"Heparin-binding assay, host cell surface binding assay with recombinant protein, competitive invasion inhibition assay, antibody-based invasion blockade, RT-PCR/Western blot expression analysis\",\n      \"journal\": \"Acta tropica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding assays with functional invasion inhibition readout; single lab\",\n      \"pmids\": [\"30664845\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A MIC3 peptide induces TNF-α production, NF-κB phosphorylation, iNOS transcription, and Ly6C expression in mouse macrophages via the TLR11/MyD88/NF-κB pathway; MIC3 can adhere to the macrophage cell membrane and enter cells; MIC3 does not induce these responses in human THP-1 macrophages (which lack TLR11); MIC3 does not induce IL-12p40 (unlike the canonical TLR11 ligand profilin), suggesting MIC3 is a distinct TLR11 ligand.\",\n      \"method\": \"MyD88 inhibitor treatment, siRNA knockdown of Tlr11, CRISPR/Cas9 Tlr11 knockout in RAW264.7 cells, NF-κB phosphorylation assay, ELISA for TNF-α, qRT-PCR for iNOS and Ly6C, immunofluorescence for MIC3 localization in macrophages\",\n      \"journal\": \"PLoS neglected tropical diseases\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three independent genetic/pharmacological methods to disrupt TLR11/MyD88 pathway (inhibitor, siRNA, CRISPR KO) with consistent results; multiple signaling readouts; cross-species (mouse vs. human) validation\",\n      \"pmids\": [\"36730424\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgMIC3 is a micronemal homodimeric adhesin that is synthesized with a pro-peptide (cleaved in a post-Golgi compartment), targeted to micronemes via signals in both its pro-peptide and EGF domains, secreted onto the parasite surface during invasion where its N-terminal chitin binding-like (CBL) domain mediates host cell receptor binding (dependent on prior pro-peptide cleavage and dimerization), shed from the surface by the rhomboid protease TgROM4, and—once secreted into the host cell environment—activates EGFR-Akt signaling (via its EGF domains) to suppress autophagic killing of the parasite, and also engages host TLR11/MyD88/NF-κB signaling to induce TNF-α and macrophage M1 polarization; MIC3 acts synergistically with MIC1 to promote virulence, and its transmembrane escorter is TgMIC8.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TgMIC3 is a micronemal secretory adhesin of Toxoplasma gondii that couples host-cell attachment during invasion to modulation of host innate immunity [#0, #9]. It is synthesized as a 40-kDa precursor and matures into a disulfide-linked homodimer of ~38-kDa polypeptides bearing five partially overlapping EGF-like domains and an N-terminal chitin binding-like (CBL) domain, and it is relocalized from the parasite apex onto the surface during cell entry [#0, #4]. Receptor binding requires two prior maturation events: cleavage of the pro-peptide to expose the CBL domain, and C-terminal-domain-mediated dimerization; the dimerization module is potent enough to convert the normally monomeric MIC8 into a receptor-competent dimer [#1]. The CBL domain engages host cell receptors, including heparan sulfate, and single substitutions that abolish CBL binding attenuate virulence, establishing adhesion as the effector activity relevant to pathogenesis [#5, #12]. Microneme targeting is encoded jointly by the pro-peptide and the EGF modules—the minimal signal being the pro-peptide plus any one EGF domain—and depends on a conserved aliphatic residue near the pro-peptide N-terminus, whereas pro-peptide cleavage, dimerization, and the CBL domain are dispensable for trafficking; the soluble adhesin reaches the micronemes as cargo of the transmembrane escorter MIC8, and is shed from the parasite surface by the rhomboid protease TgROM4 to permit productive invasion [#6, #8, #2, #7]. Beyond adhesion, secreted MIC3 acts on the host: its EGF domains activate EGFR-Akt signaling to suppress autophagic killing of the parasite, and a MIC3 peptide drives TNF-α production, NF-κB activation, iNOS and Ly6C induction, and M1 macrophage polarization through the TLR11/MyD88/NF-κB pathway [#9, #11, #13]. MIC3 acts synergistically with MIC1, and only combined mic1/mic3 disruption severely attenuates virulence [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Whether MIC3 was a structural micronemal protein or a functional adhesin was unresolved; biochemical and imaging characterization established it as a disulfide-linked homodimeric adhesin that binds host cells and redistributes to the parasite surface during invasion.\",\n      \"evidence\": \"adhesion assays with antibody blockade, affinity purification, N-terminal sequencing, invasion IFA, and cDNA cloning in T. gondii\",\n      \"pmids\": [\"11207591\", \"1944419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host receptor identity not defined\", \"Contribution of individual domains to binding not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"It was unknown what directed MIC3 to the micronemes; live-cell GFP-fusion and deletion analysis showed the cysteine-rich central region is required for proper folding and micronemal targeting, with misfolded protein defaulting to the dense-granule pathway.\",\n      \"evidence\": \"GFP-fusion imaging and deletion analysis in transfected T. gondii\",\n      \"pmids\": [\"11254953\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Minimal targeting determinant not yet defined\", \"Molecular recognition of the signal not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"How a soluble adhesin with no membrane anchor reaches the micronemes was unclear; MIC8 was shown to act as a transmembrane escorter that sorts MIC3 via cytoplasmic-tail signals and is cleaved upon discharge.\",\n      \"evidence\": \"co-localization, functional complementation, and processing assays in T. gondii\",\n      \"pmids\": [\"11861763\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct MIC3-MIC8 binding interface not mapped\", \"Stoichiometry of the escort complex unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"The requirements for MIC3 to acquire binding activity were unknown; cell-based reconstitution with truncation and domain-swap mutagenesis showed pro-peptide cleavage exposes the CBL receptor-binding site and C-terminal-domain dimerization is required for host-cell binding.\",\n      \"evidence\": \"mammalian-cell expression, MIC3-binding assays, and site-directed/domain-swap mutagenesis\",\n      \"pmids\": [\"12032066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host receptor engaged by the CBL domain not identified here\", \"Structural basis of dimer-dependent binding not solved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Whether MIC3 relocalization was coordinated with regulated microneme exocytosis was untested; quantitative imaging tied MIC3 apical loss to Ca2+-stimulated secretion accompanying invasion, with recovery ~15 min later.\",\n      \"evidence\": \"in vivo 32P labeling, quantitative IFA, stimulated-exocytosis and immunoelectron microscopy in T. gondii\",\n      \"pmids\": [\"12925131\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular trigger linking Ca2+ signaling to MIC3 discharge not defined\", \"Fate of surface MIC3 during recovery unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Whether MIC3 binding activity is functionally important in vivo was unknown; CBL point mutants and single/double knockouts established that CBL-mediated binding contributes to virulence and that MIC1 and MIC3 act synergistically, though MIC3 is dispensable for fibroblast invasion.\",\n      \"evidence\": \"single/double gene disruption, CBL-domain mutagenesis, invasion assays, and mouse virulence assays in T. gondii RH\",\n      \"pmids\": [\"15684324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of MIC1/MIC3 synergy not defined\", \"Reason for dispensability in fibroblast invasion unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The precise trafficking determinants and processing compartment were unresolved; systematic deletion and chimera analysis localized pro-peptide cleavage to a post-Golgi compartment and defined the minimal micronemal signal as pro-peptide plus any one EGF domain, distinct from the domains needed for binding.\",\n      \"evidence\": \"deletion mutants, chimeric proteins, and pulse-chase processing assays in T. gondii\",\n      \"pmids\": [\"18390648\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the pro-peptide-cleaving protease not established\", \"Basis of cell-cycle-dependent secretory retention unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"How MIC3 is removed from the surface to allow invasion was unknown; conditional ROM4 knockdown showed the rhomboid protease TgROM4 sheds MIC3, and its loss increases surface MIC3, impairs gliding and invasion despite enhanced binding.\",\n      \"evidence\": \"conditional ROM4 knockdown with adhesin-release, flow cytometry, motility and invasion assays in T. gondii RH\",\n      \"pmids\": [\"20421941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MIC3 cleavage site for ROM4 not mapped\", \"Whether MIC3 is a direct vs. indirect ROM4 substrate not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"It was unknown whether the pro-peptide targeting determinant was conserved across coccidia; mutagenesis showed pro-peptides are interchangeable across MICs and with Eimeria EtMIC5 and that a conserved aliphatic residue near the N-terminus is critical for MIC3 microneme targeting.\",\n      \"evidence\": \"N-terminal deletion/point mutagenesis and domain-swap chimeras in transfected T. gondii\",\n      \"pmids\": [\"21438967\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor recognizing the aliphatic-residue signal not identified\", \"MIC3-specific data are one component of a broader study\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Whether MIC3 has host-signaling functions beyond adhesion was unknown; knockout and recombinant-protein experiments showed its EGF domains activate EGFR-Akt signaling that blocks autophagic targeting and killing of the parasite.\",\n      \"evidence\": \"mic3 KO infection, recombinant MIC3 treatment, EGFR/Akt phosphorylation, LC3 accumulation and parasite-killing assays\",\n      \"pmids\": [\"24367261\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct MIC3-EGFR contact not demonstrated\", \"In vivo relevance of the EGFR-autophagy axis not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Host binding partners of the EGF domains beyond EGFR were unknown; a yeast two-hybrid screen identified Spata3 and Dkk2 as MIC3 interactors mediated by the tandem EGF repeats.\",\n      \"evidence\": \"yeast two-hybrid screen against a mouse library with confirmatory interaction assays\",\n      \"pmids\": [\"27053129\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence of Spata3/Dkk2 binding demonstrated in the parasite\", \"Interactions not validated in an infection context\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Whether MIC3 abundance and immunostimulatory activity relate to strain virulence was untested; iTRAQ proteomics found MIC3 ~5-fold higher in virulent RH than in TgCtwh3, and recombinant MIC3 induced TNF-α and M1 macrophage polarization.\",\n      \"evidence\": \"iTRAQ proteomics of secreted antigens and recombinant-protein macrophage stimulation with TNF-α ELISA and M1 assays\",\n      \"pmids\": [\"28000706\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor/pathway driving TNF-α and M1 polarization not defined here\", \"Causal link between MIC3 abundance and strain virulence not proven\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH; TgCtwh3\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The identity of a host receptor for the CBL/adhesin activity was still open; recombinant MIC3 was shown to bind heparan sulfate and host cell surfaces, and to competitively block invasion, with higher expression in invading parasites.\",\n      \"evidence\": \"heparin- and cell-surface binding assays, competitive and antibody-mediated invasion inhibition, and expression analysis in T. gondii RH\",\n      \"pmids\": [\"30664845\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether heparan sulfate is the physiological invasion receptor not established\", \"Domain responsible for heparin binding not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The receptor and pathway for MIC3's pro-inflammatory activity were undefined; inhibitor, siRNA and CRISPR-KO approaches showed a MIC3 peptide drives TNF-α, NF-κB, iNOS and Ly6C via TLR11/MyD88/NF-κB in mouse macrophages but not in TLR11-deficient human cells, and it is a TLR11 ligand distinct from profilin (no IL-12p40 induction).\",\n      \"evidence\": \"MyD88 inhibition, Tlr11 siRNA and CRISPR KO in RAW264.7, NF-κB/TNF-α/iNOS/Ly6C readouts, and mouse-vs-human comparison\",\n      \"pmids\": [\"36730424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct MIC3-TLR11 binding not demonstrated\", \"Human host receptor for MIC3 immunostimulation unknown\", \"Role of full-length MIC3 vs. peptide in vivo not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological host receptor engaged by the CBL domain during invasion, the direct structural basis for EGFR and TLR11 engagement, and the mechanism of MIC1/MIC3 virulence synergy remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No solved structure of MIC3 in complex with any host receptor\", \"Direct MIC3-EGFR and MIC3-TLR11 contacts not demonstrated\", \"Molecular basis of MIC1/MIC3 synergy undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0050839\", \"supporting_discovery_ids\": [0, 1, 5, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0008201\", \"supporting_discovery_ids\": [12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0042803\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0035325\", \"supporting_discovery_ids\": [13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [0, 3, 4, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0009986\", \"supporting_discovery_ids\": [0, 4, 7, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005796\", \"supporting_discovery_ids\": [6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007173\", \"supporting_discovery_ids\": [9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006914\", \"supporting_discovery_ids\": [9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0002755\", \"supporting_discovery_ids\": [13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0007165\", \"supporting_discovery_ids\": [11, 13], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [0, 5, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [\n      \"MIC3 homodimer (disulfide-linked ~38-kDa polypeptides)\",\n      \"MIC3-MIC8 escort complex\"\n    ],\n    \"partners\": [\n      \"MIC8 (transmembrane escorter)\",\n      \"MIC1 (synergistic virulence partner)\",\n      \"TgROM4 (rhomboid protease that sheds MIC3)\",\n      \"host EGFR\",\n      \"host TLR11/MyD88\",\n      \"host heparan sulfate\",\n      \"host Spata3\",\n      \"host Dkk2\"\n    ],\n    \"other_free_text\": [\n      \"Synthesized as a 40-kDa precursor with a pro-peptide cleaved in a post-Golgi compartment; pro-peptide cleavage exposes the CBL receptor-binding site.\",\n      \"Microneme targeting encoded by pro-peptide plus any one EGF domain and a conserved N-terminal aliphatic residue; pro-peptide cleavage, dimerization and CBL domain are dispensable for targeting.\",\n      \"MIC3 is a TLR11 ligand distinct from profilin (induces TNF-α but not IL-12p40) and is active in mouse but not TLR11-deficient human macrophages.\",\n      \"MIC3 deletion alone does not impair fibroblast invasion, but CBL binding mutations and combined mic1/mic3 loss attenuate virulence.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_319560","name":"MIC3","product":"microneme protein MIC3","uniprot_acc":"A0A125YUG9","chromosome":"TGME49_chrIV","start_pos":912297},{"strain":"GT1","gene_id":"TGGT1_319560","name":null,"product":"microneme protein MIC3","uniprot_acc":null,"chromosome":"TGGT1_chrIV","start_pos":856315},{"strain":"RH88","gene_id":"TGRH88_007550","name":null,"product":"microneme protein MIC3","uniprot_acc":null,"chromosome":"CM023085","start_pos":922722},{"strain":"VEG","gene_id":"TGVEG_319560","name":null,"product":"microneme protein MIC3","uniprot_acc":"V4YND8","chromosome":"TGVEG_chrIV","start_pos":911351}]}