{"gene":"PF3D7_1216700","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2014,"finding":"PLP2 (PPLP2) in P. falciparum is expressed in blood-stage schizonts and mature gametocytes, localizes to vesicular structures, and is discharged in a calcium-dependent manner upon gametocyte activation. Recombinant PLP2 has haemolytic activity toward erythrocytes. PLP2-deficient gametocytes fail to permeabilize the erythrocyte membrane (while the parasitophorous vacuole membrane ruptures normally), trapping gametocytes inside erythrocytes and reducing transmission to Anopheles. Rescue with pore-forming equinatoxin II restored exflagellation and transmission, while the pore sealant Tetronic 90R4 mimicked the PLP2-deficient phenotype in wild-type gametocytes.","method":"Gene knockout (PPLP2-deficient parasites), live imaging, calcium-dependent discharge assay, haemolytic activity assay with recombinant protein, phenotypic rescue with equinatoxin II and Tetronic 90R4, transmission assays to Anopheles mosquitoes","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with defined cellular phenotype, in vitro haemolytic assay with recombinant protein, chemical rescue experiments, multiple orthogonal methods in a single focused study","pmids":["24602217"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PLP2 (PPLP2) in P. berghei is required for erythrocyte membrane disruption during male gametocyte egress. A P. berghei PPLP2-knockout mutant displayed abnormal exflagellation, with gametocytes producing a single shared flagellum rather than eight separate flagellated gametes. Immunofluorescence and TEM confirmed that the erythrocyte membrane remained intact while the parasitophorous vacuole membrane ruptured normally. Rescue with saponin or a pore-forming toxin restored normal egress, confirming that the defect was specifically in erythrocyte membrane rupture.","method":"Gene knockout in P. berghei, immunofluorescence, transmission electron microscopy, phenotypic rescue with saponin and pore-forming toxin","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout, TEM ultrastructural analysis, chemical rescue, multiple orthogonal methods; independently corroborates findings in P. falciparum from PMID:24602217","pmids":["23461714"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2013,"finding":"PLP2 is expressed at the blood stage of P. falciparum (along with PLP1), as shown by expression analysis of perforin-like proteins in blood-stage parasites. This study primarily established the mechanism of PLP1 in Ca2+-dependent membrane permeabilization, but confirmed PLP2 expression in the blood stage.","method":"Expression analysis, blood-stage parasite culture","journal":"Nature communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — expression confirmation only for PLP2 in this paper; the mechanistic work focused on PLP1, not PLP2","pmids":["23591903"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"Secretion of PLP2 from intracellular vesicles to the periphery during gametocyte activation is dependent on the patatin-like phospholipase PfPATPL1. Conditional deletion of PfPATPL1 inhibited PLP2 secretion, reducing the efficiency of gamete egress, demonstrating that PLP2 secretion is regulated upstream by PfPATPL1-mediated vesicular discharge.","method":"Conditional gene deletion of PfPATPL1, fluorescence microscopy of PLP2 secretion, egress and exflagellation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout with defined cellular phenotype, direct imaging of PLP2 secretion; single lab, functional linkage to PLP2 secretion established","pmids":["31413195"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"AgoshRNA-mediated knockdown of PLP2 in P. berghei (using an engineered non-canonical RNAi system with Ago2) phenocopied the full gene knockout, validating PLP2's essential role and demonstrating that RNAi-based silencing can recapitulate the loss-of-function phenotype throughout the parasite life cycle.","method":"Non-canonical RNAi knockdown (Ago2 + AgoshRNA), phenotypic comparison to knockout","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic loss-of-function with defined phenotype phenocopying knockout; single lab, method validation paper with PLP2 as proof-of-concept target","pmids":["31680162"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2022,"finding":"The APCβ domain of P. vivax PLP2 (PvPLP2) binds lipid bilayers and displays tropism for negatively charged lipids enriched in the inner leaflet of the mammalian plasma membrane. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays revealed that PvPLP2 has a secondary synergistic interaction side-on from its principal bilayer interface, distinguishing it from PvPLP1 (which cannot bind lipid bilayers) and TgPLP1.","method":"Crystal structure of APCβ domain (PvPLP1), molecular dynamics simulation, site-directed mutagenesis, lipid bilayer binding functional assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — structural determination, MD simulation, mutagenesis, and functional lipid-binding assays for PvPLP2; single study","pmids":["35598848"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2026,"finding":"Cryo-EM and cryo-ET of P. vivax PLP2 (PvPLP2) reveal that it assembles on lipid bilayers into heterogeneous arc- and ring-shaped pores with variable stoichiometries. A 17-subunit pore complex was structurally determined: the pore-forming MACPF domains form the central β-barrel while peripheral APCβ domains anchor the complex to the membrane surface. A disulfide-stabilized mutant captured a pre-pore intermediate prior to membrane insertion, delineating structural transitions underlying β-barrel deployment. PvPLP2 acts preferentially on the inner leaflet of the erythrocyte membrane, driven by affinity for negatively charged lipids.","method":"Cryo-electron microscopy, cryo-electron tomography, disulfide-stabilized mutant trapping of pre-pore intermediate, lipid specificity functional assays, structural determination of 17-subunit pore complex","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM/ET structure at defined stoichiometry, mutagenesis to trap intermediate, functional lipid-binding assays; multiple orthogonal methods in a single rigorous study","pmids":["42675073"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""}]}],"current_model":"PLP2 is a MACPF/perforin-like protein that is stored in intracellular vesicles, discharged in a calcium-dependent manner (regulated upstream by PfPATPL1) upon gametocyte activation, and is specifically required for permeabilization of the erythrocyte plasma membrane (but not the parasitophorous vacuole membrane) to enable gametocyte egress; structurally, it assembles into heterogeneous arc- and ring-shaped pores on lipid bilayers through a pre-pore to pore transition in which MACPF domains form a central β-barrel anchored by peripheral APCβ domains, with preferential activity on the negatively charged inner leaflet of the erythrocyte membrane."},"narrative":{"mechanistic_narrative":"PLP2 (PF3D7_1216700) is a MACPF/perforin-like pore-forming protein required for parasite transmission, acting at gametocyte egress to permeabilize the host erythrocyte plasma membrane [PMID:24602217, PMID:23461714]. In P. falciparum, PLP2 is expressed in blood-stage schizonts and mature gametocytes, stored in vesicular structures, and discharged in a calcium-dependent manner upon gametocyte activation; recombinant PLP2 is haemolytic, and PLP2-deficient gametocytes fail to rupture the erythrocyte membrane (while the parasitophorous vacuole membrane ruptures normally), trapping gametocytes and abrogating transmission to Anopheles — a defect rescued by exogenous pore-forming toxin and phenocopied by a pore sealant [PMID:24602217]. The equivalent requirement for erythrocyte membrane disruption during male gametocyte exflagellation holds in P. berghei, where knockout mutants form a single shared flagellum and TEM confirms an intact erythrocyte membrane rescued by saponin or pore-forming toxin [PMID:23461714]. Secretion of PLP2 to the cell periphery is controlled upstream by the patatin-like phospholipase PfPATPL1, whose conditional deletion blocks PLP2 discharge and impairs gamete egress [PMID:31413195]. Structurally, PLP2 engages lipid bilayers through its APCβ domain with tropism for negatively charged lipids of the erythrocyte inner leaflet, and assembles into heterogeneous arc- and ring-shaped pores in which MACPF domains form a central β-barrel anchored by peripheral APCβ domains, transitioning from a pre-pore intermediate to the inserted pore [PMID:35598848, PMID:42675073].","teleology":[{"year":2013,"claim":"Establishing which membrane barrier blocks Plasmodium gametocyte egress required identifying a dedicated effector, and PLP2 was shown to be specifically responsible for erythrocyte membrane rupture during male gametogenesis.","evidence":"Gene knockout in P. berghei with immunofluorescence, TEM, and chemical rescue with saponin/pore-forming toxin","pmids":["23461714"],"confidence":"High","gaps":["Did not resolve how PLP2 discharge is triggered or regulated","No structural or biochemical characterization of the pore in this study"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2013,"claim":"PLP2 expression at the blood stage was confirmed alongside PLP1, situating the protein temporally even though the mechanistic focus was on the paralog PLP1.","evidence":"Expression analysis in blood-stage P. falciparum culture","pmids":["23591903"],"confidence":"Low","gaps":["Expression confirmation only; no functional data for PLP2 in this study","Mechanistic work concerned PLP1, not PLP2"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Whether PLP2 is a genuine pore-former and whether its activity underlies transmission was tested directly, showing calcium-dependent vesicular discharge, intrinsic haemolytic activity, and a transmission-blocking loss-of-function phenotype rescuable by an unrelated pore-former.","evidence":"Gene knockout, live imaging, calcium-dependent discharge and haemolytic assays with recombinant protein, chemical rescue (equinatoxin II / Tetronic 90R4), and Anopheles transmission assays in P. falciparum","pmids":["24602217"],"confidence":"High","gaps":["Molecular trigger linking calcium to discharge not defined","Pore architecture and lipid selectivity not determined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"The upstream control of PLP2 delivery was unresolved; conditional deletion of PfPATPL1 established that a patatin-like phospholipase is required for PLP2 secretion from vesicles to the cell periphery.","evidence":"Conditional deletion of PfPATPL1 with fluorescence imaging of PLP2 secretion and egress/exflagellation assays in P. falciparum","pmids":["31413195"],"confidence":"Medium","gaps":["Whether PfPATPL1 acts directly or through membrane remodeling is unresolved","Single-lab observation"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"An independent loss-of-function approach was needed to confirm PLP2's essentiality; AgoshRNA knockdown phenocopied the knockout, validating the phenotype and the silencing method.","evidence":"Non-canonical RNAi knockdown (Ago2 + AgoshRNA) in P. berghei compared to knockout","pmids":["31680162"],"confidence":"Medium","gaps":["Method-validation context rather than new mechanism","Single lab"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2022,"claim":"How PLP2 selects its target membrane was addressed by showing its APCβ domain binds lipid bilayers with tropism for negatively charged inner-leaflet lipids, distinguishing it from paralogs that cannot bind bilayers.","evidence":"Crystal structure of the APCβ domain, molecular dynamics simulation, site-directed mutagenesis, and lipid-bilayer binding assays for P. vivax PLP2","pmids":["35598848"],"confidence":"Medium","gaps":["Full-length pore assembly not resolved in this study","Single study"],"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2026,"claim":"The pore architecture and assembly pathway were unresolved until cryo-EM/ET resolved arc- and ring-shaped pores of variable stoichiometry, a defined 17-subunit complex, and a trapped pre-pore intermediate, revealing the pre-pore-to-pore β-barrel transition.","evidence":"Cryo-EM and cryo-ET, disulfide-stabilized mutant trapping the pre-pore, and lipid-specificity assays for P. vivax PLP2","pmids":["42675073"],"confidence":"High","gaps":["Structural work performed on P. vivax orthologue; in situ pore assembly on native erythrocyte membranes not directly imaged","Kinetics of pre-pore-to-pore conversion not quantified"],"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":null,"claim":"The molecular event coupling calcium signaling and PfPATPL1 activity to the timing and site of PLP2 pore assembly on the erythrocyte membrane remains undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No mechanism linking calcium-triggered discharge to membrane-side pore nucleation","No in situ structure of PLP2 pores on native gametocyte-infected erythrocyte membranes"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Homo-oligomeric arc- and ring-shaped membrane pore of variable stoichiometry, including a defined 17-subunit pore complex (P. vivax PLP2) [#6]"],"partners":["PFPATPL1 (PATATIN-LIKE PHOSPHOLIPASE) — REQUIRED UPSTREAM FOR PLP2 SECRETION [#3]"],"other_free_text":["PLP2 is required for erythrocyte plasma membrane permeabilization but not parasitophorous vacuole membrane rupture during gametocyte egress [#0, #1].","Loss-of-function is rescued by exogenous pore-forming toxins (equinatoxin II, saponin) and phenocopied by a pore sealant, confirming pore-formation is the relevant activity [#0, #1].","PLP2 preferentially targets the negatively charged inner leaflet of the erythrocyte membrane [#5, #6].","A disulfide-stabilized mutant captures a pre-pore intermediate, indicating a pre-pore-to-pore β-barrel deployment mechanism [#6]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"24602217","id":"PMC_24602217","title":"Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes.","date":"2014","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/24602217","citation_count":85,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23591903","id":"PMC_23591903","title":"Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites.","date":"2013","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/23591903","citation_count":72,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["perforin-like protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23461714","id":"PMC_23461714","title":"A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes.","date":"2013","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23461714","citation_count":72,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31413195","id":"PMC_31413195","title":"Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/31413195","citation_count":26,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfPLP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31680162","id":"PMC_31680162","title":"Gene knockdown in malaria parasites via non-canonical RNAi.","date":"2020","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/31680162","citation_count":15,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["perforin-like protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32266171","id":"PMC_32266171","title":"Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence.","date":"2020","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32266171","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"35598848","id":"PMC_35598848","title":"Structural, Functional and Computational Studies of Membrane Recognition by Plasmodium Perforin-Like Proteins 1 and 2.","date":"2022","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/35598848","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PLP2","PvPLP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"42675073","id":"PMC_42675073","title":"Molecular mechanism of pore formation by Plasmodium Perforin-like Protein 2.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/42675073","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PLP2","perforin-like protein 2","PvPLP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6793,"output_tokens":2386,"usd":0.028084,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7830,"output_tokens":3375,"usd":0.061762,"stage2_stop_reason":"end_turn"},"total_usd":0.089846,"stage1_batch_id":"msgbatch_011BPjN2BL33F8cJXD1r7V7T","stage2_batch_id":"msgbatch_01Qwg6vP7qxkpL6Gsc7tajsb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"PLP2 (PPLP2) in P. falciparum is expressed in blood-stage schizonts and mature gametocytes, localizes to vesicular structures, and is discharged in a calcium-dependent manner upon gametocyte activation. Recombinant PLP2 has haemolytic activity toward erythrocytes. PLP2-deficient gametocytes fail to permeabilize the erythrocyte membrane (while the parasitophorous vacuole membrane ruptures normally), trapping gametocytes inside erythrocytes and reducing transmission to Anopheles. Rescue with pore-forming equinatoxin II restored exflagellation and transmission, while the pore sealant Tetronic 90R4 mimicked the PLP2-deficient phenotype in wild-type gametocytes.\",\n      \"method\": \"Gene knockout (PPLP2-deficient parasites), live imaging, calcium-dependent discharge assay, haemolytic activity assay with recombinant protein, phenotypic rescue with equinatoxin II and Tetronic 90R4, transmission assays to Anopheles mosquitoes\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with defined cellular phenotype, in vitro haemolytic assay with recombinant protein, chemical rescue experiments, multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"24602217\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PLP2 (PPLP2) in P. berghei is required for erythrocyte membrane disruption during male gametocyte egress. A P. berghei PPLP2-knockout mutant displayed abnormal exflagellation, with gametocytes producing a single shared flagellum rather than eight separate flagellated gametes. Immunofluorescence and TEM confirmed that the erythrocyte membrane remained intact while the parasitophorous vacuole membrane ruptured normally. Rescue with saponin or a pore-forming toxin restored normal egress, confirming that the defect was specifically in erythrocyte membrane rupture.\",\n      \"method\": \"Gene knockout in P. berghei, immunofluorescence, transmission electron microscopy, phenotypic rescue with saponin and pore-forming toxin\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout, TEM ultrastructural analysis, chemical rescue, multiple orthogonal methods; independently corroborates findings in P. falciparum from PMID:24602217\",\n      \"pmids\": [\"23461714\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PLP2 is expressed at the blood stage of P. falciparum (along with PLP1), as shown by expression analysis of perforin-like proteins in blood-stage parasites. This study primarily established the mechanism of PLP1 in Ca2+-dependent membrane permeabilization, but confirmed PLP2 expression in the blood stage.\",\n      \"method\": \"Expression analysis, blood-stage parasite culture\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — expression confirmation only for PLP2 in this paper; the mechanistic work focused on PLP1, not PLP2\",\n      \"pmids\": [\"23591903\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Secretion of PLP2 from intracellular vesicles to the periphery during gametocyte activation is dependent on the patatin-like phospholipase PfPATPL1. Conditional deletion of PfPATPL1 inhibited PLP2 secretion, reducing the efficiency of gamete egress, demonstrating that PLP2 secretion is regulated upstream by PfPATPL1-mediated vesicular discharge.\",\n      \"method\": \"Conditional gene deletion of PfPATPL1, fluorescence microscopy of PLP2 secretion, egress and exflagellation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout with defined cellular phenotype, direct imaging of PLP2 secretion; single lab, functional linkage to PLP2 secretion established\",\n      \"pmids\": [\"31413195\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AgoshRNA-mediated knockdown of PLP2 in P. berghei (using an engineered non-canonical RNAi system with Ago2) phenocopied the full gene knockout, validating PLP2's essential role and demonstrating that RNAi-based silencing can recapitulate the loss-of-function phenotype throughout the parasite life cycle.\",\n      \"method\": \"Non-canonical RNAi knockdown (Ago2 + AgoshRNA), phenotypic comparison to knockout\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic loss-of-function with defined phenotype phenocopying knockout; single lab, method validation paper with PLP2 as proof-of-concept target\",\n      \"pmids\": [\"31680162\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The APCβ domain of P. vivax PLP2 (PvPLP2) binds lipid bilayers and displays tropism for negatively charged lipids enriched in the inner leaflet of the mammalian plasma membrane. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays revealed that PvPLP2 has a secondary synergistic interaction side-on from its principal bilayer interface, distinguishing it from PvPLP1 (which cannot bind lipid bilayers) and TgPLP1.\",\n      \"method\": \"Crystal structure of APCβ domain (PvPLP1), molecular dynamics simulation, site-directed mutagenesis, lipid bilayer binding functional assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — structural determination, MD simulation, mutagenesis, and functional lipid-binding assays for PvPLP2; single study\",\n      \"pmids\": [\"35598848\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM and cryo-ET of P. vivax PLP2 (PvPLP2) reveal that it assembles on lipid bilayers into heterogeneous arc- and ring-shaped pores with variable stoichiometries. A 17-subunit pore complex was structurally determined: the pore-forming MACPF domains form the central β-barrel while peripheral APCβ domains anchor the complex to the membrane surface. A disulfide-stabilized mutant captured a pre-pore intermediate prior to membrane insertion, delineating structural transitions underlying β-barrel deployment. PvPLP2 acts preferentially on the inner leaflet of the erythrocyte membrane, driven by affinity for negatively charged lipids.\",\n      \"method\": \"Cryo-electron microscopy, cryo-electron tomography, disulfide-stabilized mutant trapping of pre-pore intermediate, lipid specificity functional assays, structural determination of 17-subunit pore complex\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM/ET structure at defined stoichiometry, mutagenesis to trap intermediate, functional lipid-binding assays; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"42675073\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PLP2 is a MACPF/perforin-like protein that is stored in intracellular vesicles, discharged in a calcium-dependent manner (regulated upstream by PfPATPL1) upon gametocyte activation, and is specifically required for permeabilization of the erythrocyte plasma membrane (but not the parasitophorous vacuole membrane) to enable gametocyte egress; structurally, it assembles into heterogeneous arc- and ring-shaped pores on lipid bilayers through a pre-pore to pore transition in which MACPF domains form a central β-barrel anchored by peripheral APCβ domains, with preferential activity on the negatively charged inner leaflet of the erythrocyte membrane.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PLP2 (PF3D7_1216700) is a MACPF/perforin-like pore-forming protein required for parasite transmission, acting at gametocyte egress to permeabilize the host erythrocyte plasma membrane [#0, #1]. In P. falciparum, PLP2 is expressed in blood-stage schizonts and mature gametocytes, stored in vesicular structures, and discharged in a calcium-dependent manner upon gametocyte activation; recombinant PLP2 is haemolytic, and PLP2-deficient gametocytes fail to rupture the erythrocyte membrane (while the parasitophorous vacuole membrane ruptures normally), trapping gametocytes and abrogating transmission to Anopheles — a defect rescued by exogenous pore-forming toxin and phenocopied by a pore sealant [#0]. The equivalent requirement for erythrocyte membrane disruption during male gametocyte exflagellation holds in P. berghei, where knockout mutants form a single shared flagellum and TEM confirms an intact erythrocyte membrane rescued by saponin or pore-forming toxin [#1]. Secretion of PLP2 to the cell periphery is controlled upstream by the patatin-like phospholipase PfPATPL1, whose conditional deletion blocks PLP2 discharge and impairs gamete egress [#3]. Structurally, PLP2 engages lipid bilayers through its APCβ domain with tropism for negatively charged lipids of the erythrocyte inner leaflet, and assembles into heterogeneous arc- and ring-shaped pores in which MACPF domains form a central β-barrel anchored by peripheral APCβ domains, transitioning from a pre-pore intermediate to the inserted pore [#5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Establishing which membrane barrier blocks Plasmodium gametocyte egress required identifying a dedicated effector, and PLP2 was shown to be specifically responsible for erythrocyte membrane rupture during male gametogenesis.\",\n      \"evidence\": \"Gene knockout in P. berghei with immunofluorescence, TEM, and chemical rescue with saponin/pore-forming toxin\",\n      \"pmids\": [\"23461714\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how PLP2 discharge is triggered or regulated\", \"No structural or biochemical characterization of the pore in this study\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"PLP2 expression at the blood stage was confirmed alongside PLP1, situating the protein temporally even though the mechanistic focus was on the paralog PLP1.\",\n      \"evidence\": \"Expression analysis in blood-stage P. falciparum culture\",\n      \"pmids\": [\"23591903\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Expression confirmation only; no functional data for PLP2 in this study\", \"Mechanistic work concerned PLP1, not PLP2\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether PLP2 is a genuine pore-former and whether its activity underlies transmission was tested directly, showing calcium-dependent vesicular discharge, intrinsic haemolytic activity, and a transmission-blocking loss-of-function phenotype rescuable by an unrelated pore-former.\",\n      \"evidence\": \"Gene knockout, live imaging, calcium-dependent discharge and haemolytic assays with recombinant protein, chemical rescue (equinatoxin II / Tetronic 90R4), and Anopheles transmission assays in P. falciparum\",\n      \"pmids\": [\"24602217\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger linking calcium to discharge not defined\", \"Pore architecture and lipid selectivity not determined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The upstream control of PLP2 delivery was unresolved; conditional deletion of PfPATPL1 established that a patatin-like phospholipase is required for PLP2 secretion from vesicles to the cell periphery.\",\n      \"evidence\": \"Conditional deletion of PfPATPL1 with fluorescence imaging of PLP2 secretion and egress/exflagellation assays in P. falciparum\",\n      \"pmids\": [\"31413195\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PfPATPL1 acts directly or through membrane remodeling is unresolved\", \"Single-lab observation\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"An independent loss-of-function approach was needed to confirm PLP2's essentiality; AgoshRNA knockdown phenocopied the knockout, validating the phenotype and the silencing method.\",\n      \"evidence\": \"Non-canonical RNAi knockdown (Ago2 + AgoshRNA) in P. berghei compared to knockout\",\n      \"pmids\": [\"31680162\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Method-validation context rather than new mechanism\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"How PLP2 selects its target membrane was addressed by showing its APCβ domain binds lipid bilayers with tropism for negatively charged inner-leaflet lipids, distinguishing it from paralogs that cannot bind bilayers.\",\n      \"evidence\": \"Crystal structure of the APCβ domain, molecular dynamics simulation, site-directed mutagenesis, and lipid-bilayer binding assays for P. vivax PLP2\",\n      \"pmids\": [\"35598848\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full-length pore assembly not resolved in this study\", \"Single study\"],\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The pore architecture and assembly pathway were unresolved until cryo-EM/ET resolved arc- and ring-shaped pores of variable stoichiometry, a defined 17-subunit complex, and a trapped pre-pore intermediate, revealing the pre-pore-to-pore β-barrel transition.\",\n      \"evidence\": \"Cryo-EM and cryo-ET, disulfide-stabilized mutant trapping the pre-pore, and lipid-specificity assays for P. vivax PLP2\",\n      \"pmids\": [\"42675073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural work performed on P. vivax orthologue; in situ pore assembly on native erythrocyte membranes not directly imaged\", \"Kinetics of pre-pore-to-pore conversion not quantified\"],\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular event coupling calcium signaling and PfPATPL1 activity to the timing and site of PLP2 pore assembly on the erythrocyte membrane remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mechanism linking calcium-triggered discharge to membrane-side pore nucleation\", \"No in situ structure of PLP2 pores on native gametocyte-infected erythrocyte membranes\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0090729\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0019836\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0035891\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0006909\", \"supporting_discovery_ids\": []}\n    ],\n    \"complexes\": [\n      \"Homo-oligomeric arc- and ring-shaped membrane pore of variable stoichiometry, including a defined 17-subunit pore complex (P. vivax PLP2) [#6]\"\n    ],\n    \"partners\": [\n      \"PfPATPL1 (patatin-like phospholipase) — required upstream for PLP2 secretion [#3]\"\n    ],\n    \"other_free_text\": [\n      \"PLP2 is required for erythrocyte plasma membrane permeabilization but not parasitophorous vacuole membrane rupture during gametocyte egress [#0, #1].\",\n      \"Loss-of-function is rescued by exogenous pore-forming toxins (equinatoxin II, saponin) and phenocopied by a pore sealant, confirming pore-formation is the relevant activity [#0, #1].\",\n      \"PLP2 preferentially targets the negatively charged inner leaflet of the erythrocyte membrane [#5, #6].\",\n      \"A disulfide-stabilized mutant captures a pre-pore intermediate, indicating a pre-pore-to-pore β-barrel deployment mechanism [#6].\"\n    ]\n  }\n}\n```","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1216700","name":null,"product":"perforin-like protein 2","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1432400","name":null,"product":"perforin-like protein 2","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1436300","name":null,"product":"perforin-like protein 2","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"PLP2","evaluation":null}