{"gene":"PF3D7_0404400","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2007,"finding":"In P. yoelii, deletion of P36 (together with P52) causes sporozoites to fail to form a parasitophorous vacuole (PV) within hepatocytes in vitro and in vivo; p52/p36-deficient parasites localized as free entities in host cell cytoplasm or nucleoplasm and did not develop as liver stages, establishing that P36 is essential for PV formation during hepatocyte invasion.","method":"Double-gene knockout by single genetic manipulation; immunofluorescence analysis; electron microscopy; in vitro and in vivo liver-stage growth assays","journal":"Infection and immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with multiple orthogonal readouts (IFA, EM, in vitro and in vivo assays), replicated across life-cycle stages","pmids":["17517871"],"is_preprint":false,"attribution":[{"species":"Plasmodium yoelii","strain":""}]},{"year":2009,"finding":"In P. falciparum, individual or simultaneous deletion of P36 (and P52) does not affect erythrocytic replication, gametocyte production, mosquito infection, or sporozoite production, but causes parasite developmental arrest during hepatocyte infection; the double-gene deletion exhibits a more severe intrahepatocytic growth defect than either single deletion.","method":"Double-crossover recombination gene deletion; in vitro liver-stage growth assay; chimeric mouse model with human hepatocytes","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with multiple orthogonal assays, replicated in both in vitro and humanized mouse model","pmids":["19625622"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"NF54"}]},{"year":2015,"finding":"In P. yoelii/P. falciparum sporozoites, P36 engages the host hepatocyte EphA2 receptor to establish the permissive parasitophorous vacuole; deletion of P36 phenocopies loss of host EphA2, and sporozoites productively infect hepatocytes with high EphA2 expression.","method":"Genetic deletion of P36 and EphA2; hepatocyte infection assays; EphA2 expression correlation with infectivity; mouse liver infection with EphA2 knockout","journal":"Science (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (phenocopy experiment) and host receptor manipulation; direct P36–EphA2 binding not biochemically demonstrated in this abstract","pmids":["26612952"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium yoelii","strain":""}]},{"year":2017,"finding":"P36 is a major parasite determinant of host cell receptor usage during sporozoite invasion of hepatocytes: P. falciparum P36 directs use of CD81, P. vivax P36 directs use of SR-BI, while P. berghei P36 is compatible with both receptors redundantly. Genetic analysis of sporozoite factors (swapping/deleting P36 across species) established this functional link between P36 and specific host cell receptors.","method":"Genetic analysis of sporozoite P36; hepatocyte infection assays with CD81- and SR-BI-deficient cells; cross-species comparison","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis across three Plasmodium species with receptor-deficient host cells, multiple orthogonal methods","pmids":["28506360"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2018,"finding":"In P. yoelii sporozoites, P36 localizes to micronemal secretory organelles and partially co-localizes with P52. P36 and P52 form a protein complex in sporozoites (demonstrated by co-immunoprecipitation). Upon sporozoite stimulation, P36 is released as a secreted protein whereas P52 is not, supporting a model in which GPI-anchored P52 scaffolds P36 for interaction with the host cell. Single deletion of P36 alone (as well as single deletion of P52 alone) causes loss of the PV after hepatocyte invasion, phenocopying the double deletion.","method":"Live imaging of P36-mCherry tagged parasite line; co-immunoprecipitation in vivo; immunofluorescence; single-gene knockout phenotype analysis","journal":"Frontiers in cellular and infection microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, live imaging localization, multiple single-KO phenotype validations, secretion assay","pmids":["30547015"],"is_preprint":false,"attribution":[{"species":"Plasmodium yoelii","strain":""}]},{"year":2012,"finding":"The s48/45 domain structure (solved by NMR for the related Pf12 C-terminal domain) forms a β-sandwich with two sheets, conserved disulfide bonds linking the sheets, and structural homology to the SRS/SAG fold of Toxoplasma and to ephrins (ligands of Eph receptors), consistent with an adhesion function for P36 and other 6-cys family members.","method":"NMR structure determination of the Pf12 s48/45 domain; structural comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — high-quality NMR structure but performed on a paralogous domain (Pf12), not P36 itself; structural inference applied to P36 by family membership","pmids":["22493233"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"In P. berghei, Δp52&p36 parasites that do develop in Huh-7 hepatocytes (a minor fraction) do so in the absence of an apparent parasitophorous vacuole membrane, either in the cytosol or nucleus; cytosolic Δp52&p36 parasites can fully mature and produce infectious merozoites, whereas nuclear ones cannot.","method":"Immunofluorescence; electron microscopy; in vitro hepatocyte culture (Huh-7); mouse blood-stage infection assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IFA and EM with functional readout, single lab","pmids":["23227206"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2023,"finding":"The B9 protein (another 6-cys family member) interacts with P36 and P52 in a heterologous expression system, suggesting P36 is part of a multi-protein invasion complex at the sporozoite surface.","method":"Heterologous expression system protein interaction assay (pulldown/co-expression)","journal":"iScience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single pulldown in heterologous system, not confirmed in native parasite","pmids":["36761022"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"finding":"AlphaFold-based structural modeling combined with electron microscopy and small-angle X-ray scattering shows that P36 and P52 form a head-to-tail heterodimer with a conserved interaction interface across Plasmodium species. Epitope-tagged P36 variants combined with neutralization assays in P. berghei demonstrated that antibodies against epitopes on the membrane-distal side of the P36–P52 heterodimer efficiently block sporozoite invasion of hepatocytes in vitro, identifying vulnerable surface-exposed sites on P36 for antibody-mediated neutralization.","method":"AlphaFold structural modeling; electron microscopy; small-angle X-ray scattering; epitope-tagging of P36/P52; in vitro sporozoite neutralization assay in P. berghei","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — integrative structural biology (EM, SAXS, AlphaFold) plus functional neutralization experiments with tagged variants; single lab but multiple orthogonal methods","pmids":["42418521"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"P36 (PF3D7_0404400) is a micronemal, 6-cysteine domain sporozoite protein that is secreted upon hepatocyte contact and functions—in concert with GPI-anchored P52 (which acts as a scaffold)—to establish the parasitophorous vacuole during hepatocyte invasion; P36 determines which host cell receptor (CD81 for P. falciparum, SR-BI for P. vivax) is engaged during this process, likely by interacting with EphA2 and/or CD81/SR-BI on the hepatocyte surface, and P36 forms a head-to-tail heterodimeric complex with P52 whose membrane-distal face is accessible to neutralizing antibodies."},"narrative":{"mechanistic_narrative":"P36 (PF3D7_0404400) is a 6-cysteine (s48/45-domain) sporozoite protein that is essential for productive hepatocyte infection, where it enables formation of the parasitophorous vacuole (PV) in which the parasite develops as a liver stage [PMID:17517871, PMID:19625622]. In P. yoelii, P36 localizes to micronemal secretory organelles and is released as a secreted protein upon sporozoite stimulation, forming a complex with GPI-anchored P52; P52 is not itself secreted, consistent with a model in which P52 scaffolds P36 for engagement of the host cell, and single deletion of P36 alone phenocopies the PV-loss defect of the double deletion [PMID:30547015]. Genetically, P36 acts as the parasite determinant of host-cell receptor usage during invasion: P. falciparum P36 directs use of CD81, P. vivax P36 directs use of SR-BI, and P. berghei P36 is compatible with both receptors, while loss of P36 phenocopies loss of the hepatocyte EphA2 receptor [PMID:26612952, PMID:28506360]. Structurally, integrative modeling in P. berghei/P. falciparum shows P36 and P52 assemble as a head-to-tail heterodimer with a conserved interface, and antibodies targeting membrane-distal epitopes on this heterodimer block sporozoite invasion, marking P36 surface sites as neutralization-vulnerable [PMID:42418521].","teleology":[{"year":2007,"claim":"Established that P36 is required for the parasite to build the parasitophorous vacuole it needs to survive inside hepatocytes, defining its role at the invasion-to-liver-stage transition.","evidence":"Double knockout of P36/P52 in P. yoelii with IFA, EM, and in vitro/in vivo liver-stage assays","pmids":["17517871"],"confidence":"High","gaps":["Deletion was combined with P52, so P36's individual contribution was not isolated here","No molecular mechanism for how P36 promotes PV formation","Host receptor engagement not addressed"],"attribution":[{"species":"Plasmodium yoelii","strain":""}]},{"year":2009,"claim":"Extended the requirement to the human parasite and showed P36 acts specifically at hepatocyte infection without affecting other life-cycle stages, confirming a liver-stage-restricted function.","evidence":"Double-crossover deletion of P36/P52 in P. falciparum NF54 with in vitro liver assays and humanized mouse model","pmids":["19625622"],"confidence":"High","gaps":["Single-deletion phenotype less severe than double, leaving P36/P52 functional redundancy unresolved","No biochemical mechanism defined"],"attribution":[{"species":"Plasmodium falciparum","strain":"NF54"}]},{"year":2015,"claim":"Linked P36 to a specific host surface receptor, EphA2, providing the first host-side molecular partner for P36-dependent invasion.","evidence":"Genetic deletion of P36 and EphA2, hepatocyte infection assays, and EphA2 expression correlation in P. yoelii/P. falciparum","pmids":["26612952"],"confidence":"Medium","gaps":["Direct P36–EphA2 binding not biochemically demonstrated","Relationship between EphA2 and CD81/SR-BI usage not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium yoelii","strain":""}]},{"year":2017,"claim":"Showed P36 is the parasite determinant selecting which host receptor is engaged, explaining species differences in hepatocyte tropism.","evidence":"Cross-species P36 swapping/deletion with CD81- and SR-BI-deficient hepatocytes across P. falciparum, P. vivax, and P. berghei","pmids":["28506360"],"confidence":"High","gaps":["Molecular basis of P36-receptor specificity not defined","Direct binding to CD81/SR-BI not shown biochemically"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2018,"claim":"Defined P36's subcellular behavior and its physical partnership with P52, establishing a secretion/scaffolding model for host-cell engagement.","evidence":"Live imaging of P36-mCherry, in vivo co-immunoprecipitation, secretion assay, and single-KO phenotyping in P. yoelii","pmids":["30547015"],"confidence":"High","gaps":["Direct interaction with a host receptor not captured in the Co-IP","Mechanism converting secretion into PV formation unknown"],"attribution":[{"species":"Plasmodium yoelii","strain":""}]},{"year":2012,"claim":"Provided a structural rationale for P36 adhesion function via the s48/45 fold and its homology to ephrins, though shown for the paralog Pf12 rather than P36 itself.","evidence":"NMR structure of the Pf12 s48/45 domain with structural comparison to SRS/SAG folds and ephrins","pmids":["22493233"],"confidence":"Medium","gaps":["Structure solved on Pf12, not P36; applied to P36 only by family membership","No experimental structure of P36 at this stage"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"Characterized the fate of parasites that bypass P36/P52 requirement, showing PV-independent development can occur but only in the cytosol, not the nucleus.","evidence":"IFA, EM, and blood-stage infectivity readouts of Δp52&p36 P. berghei in Huh-7 cells","pmids":["23227206"],"confidence":"Medium","gaps":["P36-specific contribution not separated from P52","Why some parasites escape the PV requirement is unexplained"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2023,"claim":"Placed P36 in a larger surface invasion assembly by showing it can associate with a third 6-cys member, B9.","evidence":"Heterologous co-expression/pulldown interaction assay in P. falciparum","pmids":["36761022"],"confidence":"Low","gaps":["Single pulldown in a heterologous system, not confirmed in native parasites","Functional consequence of B9–P36 association untested","Stoichiometry and architecture of the putative complex unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"claim":"Resolved the P36–P52 assembly as a conserved head-to-tail heterodimer and mapped neutralization-vulnerable surface sites on P36, connecting structure to vaccine-relevant function.","evidence":"AlphaFold modeling, EM, SAXS, and epitope-tagged neutralization assays in P. berghei/P. falciparum","pmids":["42418521"],"confidence":"High","gaps":["No co-structure of the heterodimer bound to a host receptor","How antibody binding mechanistically blocks invasion not defined"],"attribution":[{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How P36 physically bridges its receptor-specificity function (CD81/SR-BI/EphA2) to PV membrane formation remains undefined at the biochemical level.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No demonstrated direct binding of P36 to CD81, SR-BI, or EphA2","Molecular link between receptor engagement and PV biogenesis unknown","No experimental P36 structure in a receptor-bound state"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["P36–P52 head-to-tail heterodimer [#4, #8]","Putative P36–P52–B9 invasion complex (heterologous system) [#7]"],"partners":["P52 (GPI-ANCHORED 6-CYS PARTNER/SCAFFOLD) [#4, #8]","B9 (6-CYS FAMILY MEMBER, HETEROLOGOUS INTERACTION) [#7]","HOST EPHA2 (GENETIC EPISTASIS) [#2]","HOST CD81 (P. FALCIPARUM RECEPTOR USAGE) [#3]","HOST SR-BI (P. VIVAX RECEPTOR USAGE) [#3]"],"other_free_text":["Essential for parasitophorous vacuole formation during hepatocyte invasion; loss leaves parasites free in cytoplasm/nucleoplasm [#0, #6]","Parasite determinant of host receptor specificity, species-divergent: P. falciparum P36→CD81, P. vivax P36→SR-BI, P. berghei P36 redundant for both [#3]","s48/45 β-sandwich fold with homology to ephrins inferred from paralog Pf12 structure [#5]","Membrane-distal epitopes on the P36–P52 heterodimer are targets for invasion-blocking antibodies [#8]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"19625622","id":"PMC_19625622","title":"Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design.","date":"2009","source":"Proceedings of the National Academy of Sciences of the United States of 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parasites localized as free entities in host cell cytoplasm or nucleoplasm and did not develop as liver stages, establishing that P36 is essential for PV formation during hepatocyte invasion.\",\n      \"method\": \"Double-gene knockout by single genetic manipulation; immunofluorescence analysis; electron microscopy; in vitro and in vivo liver-stage growth assays\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with multiple orthogonal readouts (IFA, EM, in vitro and in vivo assays), replicated across life-cycle stages\",\n      \"pmids\": [\"17517871\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In P. falciparum, individual or simultaneous deletion of P36 (and P52) does not affect erythrocytic replication, gametocyte production, mosquito infection, or sporozoite production, but causes parasite developmental arrest during hepatocyte infection; the double-gene deletion exhibits a more severe intrahepatocytic growth defect than either single deletion.\",\n      \"method\": \"Double-crossover recombination gene deletion; in vitro liver-stage growth assay; chimeric mouse model with human hepatocytes\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with multiple orthogonal assays, replicated in both in vitro and humanized mouse model\",\n      \"pmids\": [\"19625622\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"NF54\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In P. yoelii/P. falciparum sporozoites, P36 engages the host hepatocyte EphA2 receptor to establish the permissive parasitophorous vacuole; deletion of P36 phenocopies loss of host EphA2, and sporozoites productively infect hepatocytes with high EphA2 expression.\",\n      \"method\": \"Genetic deletion of P36 and EphA2; hepatocyte infection assays; EphA2 expression correlation with infectivity; mouse liver infection with EphA2 knockout\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (phenocopy experiment) and host receptor manipulation; direct P36–EphA2 binding not biochemically demonstrated in this abstract\",\n      \"pmids\": [\"26612952\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"P36 is a major parasite determinant of host cell receptor usage during sporozoite invasion of hepatocytes: P. falciparum P36 directs use of CD81, P. vivax P36 directs use of SR-BI, while P. berghei P36 is compatible with both receptors redundantly. Genetic analysis of sporozoite factors (swapping/deleting P36 across species) established this functional link between P36 and specific host cell receptors.\",\n      \"method\": \"Genetic analysis of sporozoite P36; hepatocyte infection assays with CD81- and SR-BI-deficient cells; cross-species comparison\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis across three Plasmodium species with receptor-deficient host cells, multiple orthogonal methods\",\n      \"pmids\": [\"28506360\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium vivax\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In P. yoelii sporozoites, P36 localizes to micronemal secretory organelles and partially co-localizes with P52. P36 and P52 form a protein complex in sporozoites (demonstrated by co-immunoprecipitation). Upon sporozoite stimulation, P36 is released as a secreted protein whereas P52 is not, supporting a model in which GPI-anchored P52 scaffolds P36 for interaction with the host cell. Single deletion of P36 alone (as well as single deletion of P52 alone) causes loss of the PV after hepatocyte invasion, phenocopying the double deletion.\",\n      \"method\": \"Live imaging of P36-mCherry tagged parasite line; co-immunoprecipitation in vivo; immunofluorescence; single-gene knockout phenotype analysis\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, live imaging localization, multiple single-KO phenotype validations, secretion assay\",\n      \"pmids\": [\"30547015\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The s48/45 domain structure (solved by NMR for the related Pf12 C-terminal domain) forms a β-sandwich with two sheets, conserved disulfide bonds linking the sheets, and structural homology to the SRS/SAG fold of Toxoplasma and to ephrins (ligands of Eph receptors), consistent with an adhesion function for P36 and other 6-cys family members.\",\n      \"method\": \"NMR structure determination of the Pf12 s48/45 domain; structural comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — high-quality NMR structure but performed on a paralogous domain (Pf12), not P36 itself; structural inference applied to P36 by family membership\",\n      \"pmids\": [\"22493233\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In P. berghei, Δp52&p36 parasites that do develop in Huh-7 hepatocytes (a minor fraction) do so in the absence of an apparent parasitophorous vacuole membrane, either in the cytosol or nucleus; cytosolic Δp52&p36 parasites can fully mature and produce infectious merozoites, whereas nuclear ones cannot.\",\n      \"method\": \"Immunofluorescence; electron microscopy; in vitro hepatocyte culture (Huh-7); mouse blood-stage infection assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IFA and EM with functional readout, single lab\",\n      \"pmids\": [\"23227206\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The B9 protein (another 6-cys family member) interacts with P36 and P52 in a heterologous expression system, suggesting P36 is part of a multi-protein invasion complex at the sporozoite surface.\",\n      \"method\": \"Heterologous expression system protein interaction assay (pulldown/co-expression)\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown in heterologous system, not confirmed in native parasite\",\n      \"pmids\": [\"36761022\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"AlphaFold-based structural modeling combined with electron microscopy and small-angle X-ray scattering shows that P36 and P52 form a head-to-tail heterodimer with a conserved interaction interface across Plasmodium species. Epitope-tagged P36 variants combined with neutralization assays in P. berghei demonstrated that antibodies against epitopes on the membrane-distal side of the P36–P52 heterodimer efficiently block sporozoite invasion of hepatocytes in vitro, identifying vulnerable surface-exposed sites on P36 for antibody-mediated neutralization.\",\n      \"method\": \"AlphaFold structural modeling; electron microscopy; small-angle X-ray scattering; epitope-tagging of P36/P52; in vitro sporozoite neutralization assay in P. berghei\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — integrative structural biology (EM, SAXS, AlphaFold) plus functional neutralization experiments with tagged variants; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"42418521\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"P36 (PF3D7_0404400) is a micronemal, 6-cysteine domain sporozoite protein that is secreted upon hepatocyte contact and functions—in concert with GPI-anchored P52 (which acts as a scaffold)—to establish the parasitophorous vacuole during hepatocyte invasion; P36 determines which host cell receptor (CD81 for P. falciparum, SR-BI for P. vivax) is engaged during this process, likely by interacting with EphA2 and/or CD81/SR-BI on the hepatocyte surface, and P36 forms a head-to-tail heterodimeric complex with P52 whose membrane-distal face is accessible to neutralizing antibodies.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"P36 (PF3D7_0404400) is a 6-cysteine (s48/45-domain) sporozoite protein that is essential for productive hepatocyte infection, where it enables formation of the parasitophorous vacuole (PV) in which the parasite develops as a liver stage [#0, #1]. In P. yoelii, P36 localizes to micronemal secretory organelles and is released as a secreted protein upon sporozoite stimulation, forming a complex with GPI-anchored P52; P52 is not itself secreted, consistent with a model in which P52 scaffolds P36 for engagement of the host cell, and single deletion of P36 alone phenocopies the PV-loss defect of the double deletion [#4]. Genetically, P36 acts as the parasite determinant of host-cell receptor usage during invasion: P. falciparum P36 directs use of CD81, P. vivax P36 directs use of SR-BI, and P. berghei P36 is compatible with both receptors, while loss of P36 phenocopies loss of the hepatocyte EphA2 receptor [#2, #3]. Structurally, integrative modeling in P. berghei/P. falciparum shows P36 and P52 assemble as a head-to-tail heterodimer with a conserved interface, and antibodies targeting membrane-distal epitopes on this heterodimer block sporozoite invasion, marking P36 surface sites as neutralization-vulnerable [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that P36 is required for the parasite to build the parasitophorous vacuole it needs to survive inside hepatocytes, defining its role at the invasion-to-liver-stage transition.\",\n      \"evidence\": \"Double knockout of P36/P52 in P. yoelii with IFA, EM, and in vitro/in vivo liver-stage assays\",\n      \"pmids\": [\"17517871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deletion was combined with P52, so P36's individual contribution was not isolated here\", \"No molecular mechanism for how P36 promotes PV formation\", \"Host receptor engagement not addressed\"],\n      \"attribution\": [{\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended the requirement to the human parasite and showed P36 acts specifically at hepatocyte infection without affecting other life-cycle stages, confirming a liver-stage-restricted function.\",\n      \"evidence\": \"Double-crossover deletion of P36/P52 in P. falciparum NF54 with in vitro liver assays and humanized mouse model\",\n      \"pmids\": [\"19625622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single-deletion phenotype less severe than double, leaving P36/P52 functional redundancy unresolved\", \"No biochemical mechanism defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"NF54\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked P36 to a specific host surface receptor, EphA2, providing the first host-side molecular partner for P36-dependent invasion.\",\n      \"evidence\": \"Genetic deletion of P36 and EphA2, hepatocyte infection assays, and EphA2 expression correlation in P. yoelii/P. falciparum\",\n      \"pmids\": [\"26612952\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct P36\\u2013EphA2 binding not biochemically demonstrated\", \"Relationship between EphA2 and CD81/SR-BI usage not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed P36 is the parasite determinant selecting which host receptor is engaged, explaining species differences in hepatocyte tropism.\",\n      \"evidence\": \"Cross-species P36 swapping/deletion with CD81- and SR-BI-deficient hepatocytes across P. falciparum, P. vivax, and P. berghei\",\n      \"pmids\": [\"28506360\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of P36-receptor specificity not defined\", \"Direct binding to CD81/SR-BI not shown biochemically\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined P36's subcellular behavior and its physical partnership with P52, establishing a secretion/scaffolding model for host-cell engagement.\",\n      \"evidence\": \"Live imaging of P36-mCherry, in vivo co-immunoprecipitation, secretion assay, and single-KO phenotyping in P. yoelii\",\n      \"pmids\": [\"30547015\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct interaction with a host receptor not captured in the Co-IP\", \"Mechanism converting secretion into PV formation unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided a structural rationale for P36 adhesion function via the s48/45 fold and its homology to ephrins, though shown for the paralog Pf12 rather than P36 itself.\",\n      \"evidence\": \"NMR structure of the Pf12 s48/45 domain with structural comparison to SRS/SAG folds and ephrins\",\n      \"pmids\": [\"22493233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure solved on Pf12, not P36; applied to P36 only by family membership\", \"No experimental structure of P36 at this stage\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Characterized the fate of parasites that bypass P36/P52 requirement, showing PV-independent development can occur but only in the cytosol, not the nucleus.\",\n      \"evidence\": \"IFA, EM, and blood-stage infectivity readouts of \\u0394p52&p36 P. berghei in Huh-7 cells\",\n      \"pmids\": [\"23227206\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"P36-specific contribution not separated from P52\", \"Why some parasites escape the PV requirement is unexplained\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed P36 in a larger surface invasion assembly by showing it can associate with a third 6-cys member, B9.\",\n      \"evidence\": \"Heterologous co-expression/pulldown interaction assay in P. falciparum\",\n      \"pmids\": [\"36761022\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single pulldown in a heterologous system, not confirmed in native parasites\", \"Functional consequence of B9\\u2013P36 association untested\", \"Stoichiometry and architecture of the putative complex unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved the P36\\u2013P52 assembly as a conserved head-to-tail heterodimer and mapped neutralization-vulnerable surface sites on P36, connecting structure to vaccine-relevant function.\",\n      \"evidence\": \"AlphaFold modeling, EM, SAXS, and epitope-tagged neutralization assays in P. berghei/P. falciparum\",\n      \"pmids\": [\"42418521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-structure of the heterodimer bound to a host receptor\", \"How antibody binding mechanistically blocks invasion not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How P36 physically bridges its receptor-specificity function (CD81/SR-BI/EphA2) to PV membrane formation remains undefined at the biochemical level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No demonstrated direct binding of P36 to CD81, SR-BI, or EphA2\", \"Molecular link between receptor engagement and PV biogenesis unknown\", \"No experimental P36 structure in a receptor-bound state\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005102\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0042802\", \"supporting_discovery_ids\": [4, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0020013\", \"supporting_discovery_ids\": [0, 1, 6]}\n    ],\n    \"complexes\": [\n      \"P36\\u2013P52 head-to-tail heterodimer [#4, #8]\",\n      \"Putative P36\\u2013P52\\u2013B9 invasion complex (heterologous system) [#7]\"\n    ],\n    \"partners\": [\n      \"P52 (GPI-anchored 6-cys partner/scaffold) [#4, #8]\",\n      \"B9 (6-cys family member, heterologous interaction) [#7]\",\n      \"Host EphA2 (genetic epistasis) [#2]\",\n      \"Host CD81 (P. falciparum receptor usage) [#3]\",\n      \"Host SR-BI (P. vivax receptor usage) [#3]\"\n    ],\n    \"other_free_text\": [\n      \"Essential for parasitophorous vacuole formation during hepatocyte invasion; loss leaves parasites free in cytoplasm/nucleoplasm [#0, #6]\",\n      \"Parasite determinant of host receptor specificity, species-divergent: P. falciparum P36\\u2192CD81, P. vivax P36\\u2192SR-BI, P. berghei P36 redundant for both [#3]\",\n      \"s48/45 \\u03b2-sandwich fold with homology to ephrins inferred from paralog Pf12 structure [#5]\",\n      \"Membrane-distal epitopes on the P36\\u2013P52 heterodimer are targets for invasion-blocking antibodies [#8]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0404400","name":null,"product":"6-cysteine protein P36","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1002100","name":null,"product":"6-cysteine protein P36","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0302400","name":null,"product":"6-cysteine protein P36, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"P36","evaluation":null}