{"gene":"PF3D7_0405300","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2012,"finding":"LISP2 (liver-specific protein 2) is expressed in Plasmodium berghei liver-stage parasites starting 24 h post-infection, localizes to the parasitophorous vacuole, and is subsequently exported to the cytoplasm and nucleus of host hepatocytes. Gene targeting (knockout) of LISP2 caused the majority of liver-stage parasites to arrest during merozoite formation, establishing a required role in parasite-host interactions during intrahepatic development.","method":"Immunofluorescence staining with anti-LISP2 antibodies; gene targeting/disruption with phenotypic analysis of liver-stage development","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal localization by immunofluorescence plus genetic loss-of-function with specific developmental arrest phenotype, single lab but multiple orthogonal methods","pmids":["23216750"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2019,"finding":"LISP2 is expressed in early developing liver-stage parasites (schizonts) but not in dormant hypnozoites of P. cynomolgi (in vitro) and P. vivax (in vivo), establishing LISP2 as a molecular marker that discriminates active liver-stage schizogony from dormancy. Prophylactic drugs selectively killed all LISP2-positive parasites, while LISP2-negative hypnozoites were only sensitive to tafenoquine, functionally linking LISP2 expression to the onset of schizogony.","method":"Immunofluorescence analysis of hepatocytes infected with P. cynomolgi (in vitro) and P. vivax (in vivo); drug treatment experiments correlating LISP2 status with drug sensitivity","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct immunofluorescence localization with functional drug-sensitivity validation in two relapsing-malaria species, multiple orthogonal readouts","pmids":["31094679"],"is_preprint":false,"attribution":[{"species":"Plasmodium cynomolgi","strain":""},{"species":"Plasmodium vivax","strain":""}]},{"year":2018,"finding":"Dual deletion of LISP2 and PlasMei2 in Plasmodium yoelii produced a synthetic lethal phenotype causing complete late liver-stage arrest with no breakthrough blood-stage infection, establishing that LISP2 acts in a pathway required for late liver-stage development and that its function is partially redundant with PlasMei2 in this context.","method":"Genetic double-knockout (LISP2 and PlasMei2) in P. yoelii with assessment of liver-stage arrest and blood-stage breakthrough by in vivo infection of mice","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via double-knockout with defined arrest phenotype, single lab","pmids":["29440367"],"is_preprint":false,"attribution":[{"species":"Plasmodium yoelii","strain":""}]},{"year":2016,"finding":"Single deletion of LISP2 in Plasmodium berghei ANKA caused liver-stage developmental arrest but with occasional breakthrough infections; combining LISP2 deletion with uis3 deletion produced complete attenuation, indicating LISP2 function is required for liver-stage completion and that its pathway is partially separable from that of UIS3.","method":"Single and double gene knockout in P. berghei with in vivo sporozoite challenge assays measuring breakthrough blood-stage infection","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with epistasis, single lab, in vivo phenotypic readout","pmids":["27241521"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":"ANKA"}]},{"year":2015,"finding":"The LISP2 gene promoter region from Plasmodium berghei confers liver-stage-specific (stage-enriched) expression in vivo, as confirmed by dual-luminescence reporter assays. Substitution and deletion analyses identified specific elements within the promoter required for stage-specific activity.","method":"Dual luminescence reporter assays with promoter substitution and deletion analyses in P. berghei in vivo and in vitro","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter functional dissection with multiple deletion/substitution constructs, single lab","pmids":["25874388"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"finding":"The LISP2 promoter (schizont-specific) was used to drive firefly luciferase expression in transgenic P. cynomolgi, enabling bioluminescence-based discrimination of liver-stage schizonts from hypnozoites. This functional use confirms that the LISP2 promoter is active specifically in developing (schizont) liver stages but not in dormant hypnozoites of P. cynomolgi.","method":"Transgenic parasite engineering with firefly luciferase under lisp2 promoter control; bioluminescence assay in 96-well and 384-well formats","journal":"Analytical chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct functional use of LISP2 promoter in transgenic parasites with bioluminescence readout, single lab","pmids":["32267675"],"is_preprint":false,"attribution":[{"species":"Plasmodium cynomolgi","strain":""}]},{"year":2022,"finding":"Post-invasion knockdown of EXP2 in Plasmodium berghei largely abolished expression of the mid-to-late liver-stage marker LISP2, placing LISP2 expression downstream of EXP2/PTEX function during intrahepatic development. This establishes that LISP2 accumulation depends on intact EXP2-mediated vacuolar pore activity.","method":"glmS ribozyme-mediated conditional knockdown of EXP2 after hepatocyte invasion in P. berghei; immunofluorescence to assess LISP2 expression; nanoluciferase reporter for developmental readout","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockdown with two orthogonal readouts (immunofluorescence for LISP2, nanoluciferase for development), single lab","pmids":["36445080"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"finding":"P. falciparum LISP2 (Pf-LISP2) contains a classical bipartite nuclear localization signal (NLS) in its C-terminal region that is essential for nuclear import into HepG2 hepatocytes. Fusion of the Pf-LISP2 NLS to Pf-aldolase directed the chimera exclusively to the nucleus. Mutation of key lysine and arginine residues in the bipartite NLS abolished nuclear localization. Nuclear import required importin-α/β interaction, as coexpression with the importin-α/β inhibitor mCherry-Bimax2 blocked translocation. Pf-LISP2 lacks any nuclear export signal. Phylogenetic analysis showed this NLS is unique to P. falciparum and P. reichenowi.","method":"NLS-fusion constructs (Pf-aldolase-NLS-EGFP) expressed in HepG2 cells; site-directed mutagenesis of NLS basic residues; importin-α/β inhibitor (mCherry-Bimax2) coexpression; fluorescence microscopy; phylogenetic analysis","journal":"ACS infectious diseases","confidence":"High","confidence_rationale":"Tier 1 / Moderate — functional reconstitution with NLS-fusion constructs, mutagenesis of active residues, and pharmacological inhibition of importin, all in a single study with multiple orthogonal approaches","pmids":["39601512"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"finding":"P. falciparum LISP2 localizes to the periphery of P. falciparum liver stages in humanized mice but is not exported beyond the parasitophorous vacuole membrane (PVM). Disruption of PTEX members PTEX150 and EXP2 reduced LISP2 expression in P. falciparum liver stages, indicating that LISP2 expression or accumulation is dependent — directly or indirectly — on intact PTEX function.","method":"Flp/FRT conditional gene excision of ptex150 and exp2 in P. falciparum sporozoites; localization of LISP2 by immunofluorescence in humanized mice liver stages; comparison of LISP2 expression between knockout and wild-type parasites","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional genetic disruption with direct protein localization, single lab, in vivo humanized mouse model","pmids":["38968107"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"LISP2 (PF3D7_0405300) is a liver-stage-specific secreted protein that is expressed in developing (but not dormant) intrahepatic parasites, is trafficked through the parasitophorous vacuole and exported to the hepatocyte cytoplasm and nucleus; in P. falciparum, nuclear import is mediated by a bipartite NLS in its C-terminal region via the importin-α/β pathway, and genetic loss-of-function across multiple Plasmodium species consistently causes late liver-stage developmental arrest during merozoite formation, while its expression and peripheral vacuolar localization depend partly on the PTEX translocon components EXP2 and PTEX150."},"narrative":{"mechanistic_narrative":"LISP2 (PF3D7_0405300) is a liver-stage-specific protein whose expression marks the transition of intrahepatic Plasmodium parasites into active schizogony and whose function is required for completion of liver-stage development [PMID:23216750, PMID:31094679]. First characterized in P. berghei, LISP2 is expressed from ~24 h post-infection, localizes to the parasitophorous vacuole, and in rodent parasites is exported to the cytoplasm and nucleus of the host hepatocyte; its genetic disruption arrests the majority of parasites during merozoite formation [PMID:23216750]. Across relapsing-malaria species (P. cynomolgi, P. vivax), LISP2 is expressed in developing schizonts but not in dormant hypnozoites, making it a molecular and functional marker that discriminates active liver-stage development from dormancy and correlating its expression with drug-sensitive schizogony [PMID:31094679]. Genetic loss-of-function is consistent across Plasmodium species, with residual breakthrough after single deletion in P. berghei that becomes complete attenuation when combined with uis3 deletion, and synthetic lethality with PlasMei2 deletion in P. yoelii, placing LISP2 in a late liver-stage developmental pathway that is partially redundant with these factors [PMID:29440367, PMID:27241521]. In P. falciparum, LISP2 carries a classical bipartite nuclear localization signal in its C-terminus that is necessary and sufficient to direct nuclear import into hepatocytes via the importin-α/β pathway, while the protein lacks any export signal [PMID:39601512]; notably, in P. falciparum humanized-mouse liver stages LISP2 remains at the parasite periphery and is not exported beyond the parasitophorous vacuole membrane, and its accumulation depends on the PTEX translocon components EXP2 and PTEX150 [PMID:38968107, PMID:36445080]. The LISP2 promoter itself is a functional, stage-specific regulatory element sufficient to drive schizont-restricted reporter expression in vivo [PMID:25874388, PMID:32267675]. Beyond these findings, no biochemical activity or direct molecular partners for the LISP2 protein have been characterized in the available corpus.","teleology":[{"year":2012,"claim":"Established that LISP2 is a liver-stage-expressed protein exported from the vacuole to the host hepatocyte and required for intrahepatic development, defining its cellular role in parasite-host interaction.","evidence":"Anti-LISP2 immunofluorescence plus gene knockout with liver-stage phenotyping in P. berghei","pmids":["23216750"],"confidence":"High","gaps":["No molecular mechanism for the arrest during merozoite formation","Host-cell targets or biochemical activity of exported LISP2 unknown"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2015,"claim":"Dissected the LISP2 promoter to show it is an autonomous stage-specific regulatory element, explaining how LISP2 expression is restricted to the liver stage.","evidence":"Dual-luminescence reporter assays with promoter substitution/deletion constructs in P. berghei","pmids":["25874388"],"confidence":"Medium","gaps":["Trans-acting factors binding the identified promoter elements not identified","Does not connect promoter activity to protein function"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2016,"claim":"Positioned LISP2 in a partially redundant late liver-stage pathway by showing single deletion allows breakthrough while combination with uis3 deletion gives complete attenuation.","evidence":"Single and double gene knockout with in vivo sporozoite challenge in P. berghei ANKA","pmids":["27241521"],"confidence":"Medium","gaps":["Molecular basis of redundancy with UIS3 not defined","Whether LISP2 and UIS3 act in the same biochemical process unclear"],"attribution":[{"species":"Plasmodium berghei","strain":"ANKA"}]},{"year":2018,"claim":"Demonstrated genetic synthetic lethality between LISP2 and PlasMei2, establishing that LISP2 function overlaps with PlasMei2 in enabling late liver-stage development.","evidence":"Double gene knockout with in vivo mouse infection and blood-stage breakthrough assessment in P. yoelii","pmids":["29440367"],"confidence":"Medium","gaps":["Mechanistic link between LISP2 and PlasMei2 not defined","Single lab; epistasis not extended to other species"],"attribution":[{"species":"Plasmodium yoelii","strain":""}]},{"year":2019,"claim":"Defined LISP2 as a marker discriminating active schizogony from dormant hypnozoites, functionally linking its expression to the onset of development and drug sensitivity.","evidence":"Immunofluorescence in P. cynomolgi (in vitro) and P. vivax (in vivo) with drug-sensitivity correlation","pmids":["31094679"],"confidence":"High","gaps":["Regulatory switch controlling LISP2 onset at schizogony not identified","Whether LISP2 is causal for, or merely coincident with, the dormancy exit unknown"],"attribution":[{"species":"Plasmodium cynomolgi","strain":""},{"species":"Plasmodium vivax","strain":""}]},{"year":2020,"claim":"Exploited the LISP2 promoter as a schizont-specific driver, confirming it is active in developing but not dormant liver stages and providing a bioluminescent readout for schizogony.","evidence":"Transgenic P. cynomolgi expressing luciferase under the lisp2 promoter with bioluminescence assays","pmids":["32267675"],"confidence":"Medium","gaps":["Applied use rather than new mechanistic insight into LISP2 protein","Promoter regulation in P. cynomolgi not dissected at element level"],"attribution":[{"species":"Plasmodium cynomolgi","strain":""}]},{"year":2022,"claim":"Placed LISP2 expression downstream of PTEX pore activity by showing EXP2 knockdown abolishes LISP2 accumulation during intrahepatic development.","evidence":"glmS conditional EXP2 knockdown with LISP2 immunofluorescence and nanoluciferase developmental readout in P. berghei","pmids":["36445080"],"confidence":"Medium","gaps":["Whether EXP2 acts on LISP2 directly or via general developmental arrest unresolved","Does not distinguish effects on transcription vs trafficking/stability"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"claim":"Resolved the mechanism of LISP2 nuclear targeting by identifying a C-terminal bipartite NLS necessary and sufficient for importin-α/β-mediated import into hepatocytes.","evidence":"NLS-fusion constructs, site-directed mutagenesis, and importin inhibitor coexpression in HepG2 cells with fluorescence microscopy in P. falciparum","pmids":["39601512"],"confidence":"High","gaps":["Nuclear function of imported LISP2 not established","NLS shown unique to P. falciparum/P. reichenowi, so import route may differ in rodent species"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"claim":"Showed that P. falciparum LISP2 stays at the parasite periphery (not exported beyond the PVM) and that its expression depends on PTEX components PTEX150 and EXP2, refining LISP2 trafficking and its dependence on the translocon.","evidence":"Flp/FRT conditional excision of ptex150 and exp2 with LISP2 immunofluorescence in humanized-mouse P. falciparum liver stages","pmids":["38968107"],"confidence":"Medium","gaps":["Peripheral localization in P. falciparum contrasts with host-cytoplasm/nucleus export reported in P. berghei; species difference unresolved","Whether PTEX dependence reflects direct export of LISP2 or indirect developmental effect unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The biochemical activity of the LISP2 protein and its molecular targets in the parasite or host cell remain unknown, leaving open how LISP2 drives liver-stage completion and what it does once imported into the hepatocyte nucleus.","evidence":"No discovery in the corpus assigns a catalytic activity, binding partner, or nuclear target to LISP2","pmids":[],"confidence":"Low","gaps":["No direct protein interaction partner identified","No enzymatic or structural characterization","Function of nuclear-imported LISP2 in host hepatocytes undefined"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["IMPORTIN-Α/Β (MEDIATES LISP2 NUCLEAR IMPORT IN P. FALCIPARUM) [#7]","EXP2 / PTEX150 (PTEX TRANSLOCON; LISP2 EXPRESSION/ACCUMULATION DEPENDS ON THEM) [#6, #8]","PLASMEI2 (SYNTHETIC-LETHAL GENETIC INTERACTION IN P. YOELII) [#2]","UIS3 (GENETIC INTERACTION; COMBINED DELETION GIVES COMPLETE ATTENUATION IN P. BERGHEI) [#3]"],"other_free_text":["LISP2 is expressed in developing liver-stage schizonts but not in dormant hypnozoites, serving as a marker of active schizogony [#1, #5].","The LISP2 promoter is an autonomous liver-stage/schizont-specific regulatory element usable to drive stage-restricted reporter expression [#4, #5].","Genetic loss-of-function causes late liver-stage developmental arrest during merozoite formation across multiple Plasmodium species [#0, #2, #3].","Species divergence: exported to host hepatocyte cytoplasm and nucleus in P. berghei [#0], but retained at the parasite periphery (not exported beyond the PVM) in P. falciparum liver stages [#8].","The C-terminal bipartite NLS is phylogenetically unique to P. falciparum and P. reichenowi [#7]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"23216750","id":"PMC_23216750","title":"Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation.","date":"2012","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23216750","citation_count":68,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31094679","id":"PMC_31094679","title":"The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31094679","citation_count":46,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29440367","id":"PMC_29440367","title":"A Plasmodium Parasite with Complete Late Liver Stage Arrest Protects against Preerythrocytic and Erythrocytic Stage Infection in Mice.","date":"2018","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/29440367","citation_count":37,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27241521","id":"PMC_27241521","title":"Protective efficacy and safety of liver stage attenuated malaria parasites.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27241521","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24691399","id":"PMC_24691399","title":"D-Glucose concentration is the key factor facilitating liver stage maturation of Plasmodium.","date":"2014","source":"Parasitology international","url":"https://pubmed.ncbi.nlm.nih.gov/24691399","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26348884","id":"PMC_26348884","title":"In silico identification of genetically attenuated vaccine candidate genes for Plasmodium liver stage.","date":"2015","source":"Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/26348884","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25874388","id":"PMC_25874388","title":"In vivo and in vitro characterization of a Plasmodium liver stage-specific promoter.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25874388","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32267675","id":"PMC_32267675","title":"Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Antirelapse Compounds.","date":"2020","source":"Analytical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32267675","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36445080","id":"PMC_36445080","title":"The PTEX Pore Component EXP2 Is Important for Intrahepatic Development during the Plasmodium Liver Stage.","date":"2022","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/36445080","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38968107","id":"PMC_38968107","title":"Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development.","date":"2024","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/38968107","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"33228734","id":"PMC_33228734","title":"Definition of constitutive and stage-enriched promoters in the rodent malaria parasite, Plasmodium yoelii.","date":"2020","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/33228734","citation_count":5,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"41023197","id":"PMC_41023197","title":"Complete attenuation of Plasmodium falciparum sporozoites by atovaquone-proguanil.","date":"2025","source":"EMBO molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41023197","citation_count":2,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"39601512","id":"PMC_39601512","title":"Mapping of Nuclear Localization Signal in Secreted Liver-Specific Protein 2 of Plasmodium falciparum.","date":"2024","source":"ACS infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/39601512","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2","PfLISP2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"41972120","id":"PMC_41972120","title":"Naturally acquired antibodies to Plasmodium vivax Pv_LISP-2, a potential liver stage vaccine antigen.","date":"2026","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41972120","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LISP2","liver specific protein 2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9817,"output_tokens":2845,"usd":0.036063,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8479,"output_tokens":4083,"usd":0.072235,"stage2_stop_reason":"end_turn"},"total_usd":0.108298,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"LISP2 (liver-specific protein 2) is expressed in Plasmodium berghei liver-stage parasites starting 24 h post-infection, localizes to the parasitophorous vacuole, and is subsequently exported to the cytoplasm and nucleus of host hepatocytes. Gene targeting (knockout) of LISP2 caused the majority of liver-stage parasites to arrest during merozoite formation, establishing a required role in parasite-host interactions during intrahepatic development.\",\n      \"method\": \"Immunofluorescence staining with anti-LISP2 antibodies; gene targeting/disruption with phenotypic analysis of liver-stage development\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal localization by immunofluorescence plus genetic loss-of-function with specific developmental arrest phenotype, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"23216750\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LISP2 is expressed in early developing liver-stage parasites (schizonts) but not in dormant hypnozoites of P. cynomolgi (in vitro) and P. vivax (in vivo), establishing LISP2 as a molecular marker that discriminates active liver-stage schizogony from dormancy. Prophylactic drugs selectively killed all LISP2-positive parasites, while LISP2-negative hypnozoites were only sensitive to tafenoquine, functionally linking LISP2 expression to the onset of schizogony.\",\n      \"method\": \"Immunofluorescence analysis of hepatocytes infected with P. cynomolgi (in vitro) and P. vivax (in vivo); drug treatment experiments correlating LISP2 status with drug sensitivity\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct immunofluorescence localization with functional drug-sensitivity validation in two relapsing-malaria species, multiple orthogonal readouts\",\n      \"pmids\": [\"31094679\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium cynomolgi\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Dual deletion of LISP2 and PlasMei2 in Plasmodium yoelii produced a synthetic lethal phenotype causing complete late liver-stage arrest with no breakthrough blood-stage infection, establishing that LISP2 acts in a pathway required for late liver-stage development and that its function is partially redundant with PlasMei2 in this context.\",\n      \"method\": \"Genetic double-knockout (LISP2 and PlasMei2) in P. yoelii with assessment of liver-stage arrest and blood-stage breakthrough by in vivo infection of mice\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via double-knockout with defined arrest phenotype, single lab\",\n      \"pmids\": [\"29440367\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Single deletion of LISP2 in Plasmodium berghei ANKA caused liver-stage developmental arrest but with occasional breakthrough infections; combining LISP2 deletion with uis3 deletion produced complete attenuation, indicating LISP2 function is required for liver-stage completion and that its pathway is partially separable from that of UIS3.\",\n      \"method\": \"Single and double gene knockout in P. berghei with in vivo sporozoite challenge assays measuring breakthrough blood-stage infection\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with epistasis, single lab, in vivo phenotypic readout\",\n      \"pmids\": [\"27241521\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"ANKA\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The LISP2 gene promoter region from Plasmodium berghei confers liver-stage-specific (stage-enriched) expression in vivo, as confirmed by dual-luminescence reporter assays. Substitution and deletion analyses identified specific elements within the promoter required for stage-specific activity.\",\n      \"method\": \"Dual luminescence reporter assays with promoter substitution and deletion analyses in P. berghei in vivo and in vitro\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter functional dissection with multiple deletion/substitution constructs, single lab\",\n      \"pmids\": [\"25874388\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The LISP2 promoter (schizont-specific) was used to drive firefly luciferase expression in transgenic P. cynomolgi, enabling bioluminescence-based discrimination of liver-stage schizonts from hypnozoites. This functional use confirms that the LISP2 promoter is active specifically in developing (schizont) liver stages but not in dormant hypnozoites of P. cynomolgi.\",\n      \"method\": \"Transgenic parasite engineering with firefly luciferase under lisp2 promoter control; bioluminescence assay in 96-well and 384-well formats\",\n      \"journal\": \"Analytical chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct functional use of LISP2 promoter in transgenic parasites with bioluminescence readout, single lab\",\n      \"pmids\": [\"32267675\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium cynomolgi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Post-invasion knockdown of EXP2 in Plasmodium berghei largely abolished expression of the mid-to-late liver-stage marker LISP2, placing LISP2 expression downstream of EXP2/PTEX function during intrahepatic development. This establishes that LISP2 accumulation depends on intact EXP2-mediated vacuolar pore activity.\",\n      \"method\": \"glmS ribozyme-mediated conditional knockdown of EXP2 after hepatocyte invasion in P. berghei; immunofluorescence to assess LISP2 expression; nanoluciferase reporter for developmental readout\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockdown with two orthogonal readouts (immunofluorescence for LISP2, nanoluciferase for development), single lab\",\n      \"pmids\": [\"36445080\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"P. falciparum LISP2 (Pf-LISP2) contains a classical bipartite nuclear localization signal (NLS) in its C-terminal region that is essential for nuclear import into HepG2 hepatocytes. Fusion of the Pf-LISP2 NLS to Pf-aldolase directed the chimera exclusively to the nucleus. Mutation of key lysine and arginine residues in the bipartite NLS abolished nuclear localization. Nuclear import required importin-α/β interaction, as coexpression with the importin-α/β inhibitor mCherry-Bimax2 blocked translocation. Pf-LISP2 lacks any nuclear export signal. Phylogenetic analysis showed this NLS is unique to P. falciparum and P. reichenowi.\",\n      \"method\": \"NLS-fusion constructs (Pf-aldolase-NLS-EGFP) expressed in HepG2 cells; site-directed mutagenesis of NLS basic residues; importin-α/β inhibitor (mCherry-Bimax2) coexpression; fluorescence microscopy; phylogenetic analysis\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — functional reconstitution with NLS-fusion constructs, mutagenesis of active residues, and pharmacological inhibition of importin, all in a single study with multiple orthogonal approaches\",\n      \"pmids\": [\"39601512\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"P. falciparum LISP2 localizes to the periphery of P. falciparum liver stages in humanized mice but is not exported beyond the parasitophorous vacuole membrane (PVM). Disruption of PTEX members PTEX150 and EXP2 reduced LISP2 expression in P. falciparum liver stages, indicating that LISP2 expression or accumulation is dependent — directly or indirectly — on intact PTEX function.\",\n      \"method\": \"Flp/FRT conditional gene excision of ptex150 and exp2 in P. falciparum sporozoites; localization of LISP2 by immunofluorescence in humanized mice liver stages; comparison of LISP2 expression between knockout and wild-type parasites\",\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 genetic disruption with direct protein localization, single lab, in vivo humanized mouse model\",\n      \"pmids\": [\"38968107\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"LISP2 (PF3D7_0405300) is a liver-stage-specific secreted protein that is expressed in developing (but not dormant) intrahepatic parasites, is trafficked through the parasitophorous vacuole and exported to the hepatocyte cytoplasm and nucleus; in P. falciparum, nuclear import is mediated by a bipartite NLS in its C-terminal region via the importin-α/β pathway, and genetic loss-of-function across multiple Plasmodium species consistently causes late liver-stage developmental arrest during merozoite formation, while its expression and peripheral vacuolar localization depend partly on the PTEX translocon components EXP2 and PTEX150.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LISP2 (PF3D7_0405300) is a liver-stage-specific protein whose expression marks the transition of intrahepatic Plasmodium parasites into active schizogony and whose function is required for completion of liver-stage development [#0, #1]. First characterized in P. berghei, LISP2 is expressed from ~24 h post-infection, localizes to the parasitophorous vacuole, and in rodent parasites is exported to the cytoplasm and nucleus of the host hepatocyte; its genetic disruption arrests the majority of parasites during merozoite formation [#0]. Across relapsing-malaria species (P. cynomolgi, P. vivax), LISP2 is expressed in developing schizonts but not in dormant hypnozoites, making it a molecular and functional marker that discriminates active liver-stage development from dormancy and correlating its expression with drug-sensitive schizogony [#1]. Genetic loss-of-function is consistent across Plasmodium species, with residual breakthrough after single deletion in P. berghei that becomes complete attenuation when combined with uis3 deletion, and synthetic lethality with PlasMei2 deletion in P. yoelii, placing LISP2 in a late liver-stage developmental pathway that is partially redundant with these factors [#2, #3]. In P. falciparum, LISP2 carries a classical bipartite nuclear localization signal in its C-terminus that is necessary and sufficient to direct nuclear import into hepatocytes via the importin-\\u03b1/\\u03b2 pathway, while the protein lacks any export signal [#7]; notably, in P. falciparum humanized-mouse liver stages LISP2 remains at the parasite periphery and is not exported beyond the parasitophorous vacuole membrane, and its accumulation depends on the PTEX translocon components EXP2 and PTEX150 [#8, #6]. The LISP2 promoter itself is a functional, stage-specific regulatory element sufficient to drive schizont-restricted reporter expression in vivo [#4, #5]. Beyond these findings, no biochemical activity or direct molecular partners for the LISP2 protein have been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that LISP2 is a liver-stage-expressed protein exported from the vacuole to the host hepatocyte and required for intrahepatic development, defining its cellular role in parasite-host interaction.\",\n      \"evidence\": \"Anti-LISP2 immunofluorescence plus gene knockout with liver-stage phenotyping in P. berghei\",\n      \"pmids\": [\"23216750\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No molecular mechanism for the arrest during merozoite formation\", \"Host-cell targets or biochemical activity of exported LISP2 unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Dissected the LISP2 promoter to show it is an autonomous stage-specific regulatory element, explaining how LISP2 expression is restricted to the liver stage.\",\n      \"evidence\": \"Dual-luminescence reporter assays with promoter substitution/deletion constructs in P. berghei\",\n      \"pmids\": [\"25874388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trans-acting factors binding the identified promoter elements not identified\", \"Does not connect promoter activity to protein function\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Positioned LISP2 in a partially redundant late liver-stage pathway by showing single deletion allows breakthrough while combination with uis3 deletion gives complete attenuation.\",\n      \"evidence\": \"Single and double gene knockout with in vivo sporozoite challenge in P. berghei ANKA\",\n      \"pmids\": [\"27241521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of redundancy with UIS3 not defined\", \"Whether LISP2 and UIS3 act in the same biochemical process unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"ANKA\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated genetic synthetic lethality between LISP2 and PlasMei2, establishing that LISP2 function overlaps with PlasMei2 in enabling late liver-stage development.\",\n      \"evidence\": \"Double gene knockout with in vivo mouse infection and blood-stage breakthrough assessment in P. yoelii\",\n      \"pmids\": [\"29440367\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between LISP2 and PlasMei2 not defined\", \"Single lab; epistasis not extended to other species\"],\n      \"attribution\": [{\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined LISP2 as a marker discriminating active schizogony from dormant hypnozoites, functionally linking its expression to the onset of development and drug sensitivity.\",\n      \"evidence\": \"Immunofluorescence in P. cynomolgi (in vitro) and P. vivax (in vivo) with drug-sensitivity correlation\",\n      \"pmids\": [\"31094679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulatory switch controlling LISP2 onset at schizogony not identified\", \"Whether LISP2 is causal for, or merely coincident with, the dormancy exit unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium cynomolgi\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Exploited the LISP2 promoter as a schizont-specific driver, confirming it is active in developing but not dormant liver stages and providing a bioluminescent readout for schizogony.\",\n      \"evidence\": \"Transgenic P. cynomolgi expressing luciferase under the lisp2 promoter with bioluminescence assays\",\n      \"pmids\": [\"32267675\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Applied use rather than new mechanistic insight into LISP2 protein\", \"Promoter regulation in P. cynomolgi not dissected at element level\"],\n      \"attribution\": [{\"species\": \"Plasmodium cynomolgi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed LISP2 expression downstream of PTEX pore activity by showing EXP2 knockdown abolishes LISP2 accumulation during intrahepatic development.\",\n      \"evidence\": \"glmS conditional EXP2 knockdown with LISP2 immunofluorescence and nanoluciferase developmental readout in P. berghei\",\n      \"pmids\": [\"36445080\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EXP2 acts on LISP2 directly or via general developmental arrest unresolved\", \"Does not distinguish effects on transcription vs trafficking/stability\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the mechanism of LISP2 nuclear targeting by identifying a C-terminal bipartite NLS necessary and sufficient for importin-\\u03b1/\\u03b2-mediated import into hepatocytes.\",\n      \"evidence\": \"NLS-fusion constructs, site-directed mutagenesis, and importin inhibitor coexpression in HepG2 cells with fluorescence microscopy in P. falciparum\",\n      \"pmids\": [\"39601512\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear function of imported LISP2 not established\", \"NLS shown unique to P. falciparum/P. reichenowi, so import route may differ in rodent species\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed that P. falciparum LISP2 stays at the parasite periphery (not exported beyond the PVM) and that its expression depends on PTEX components PTEX150 and EXP2, refining LISP2 trafficking and its dependence on the translocon.\",\n      \"evidence\": \"Flp/FRT conditional excision of ptex150 and exp2 with LISP2 immunofluorescence in humanized-mouse P. falciparum liver stages\",\n      \"pmids\": [\"38968107\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Peripheral localization in P. falciparum contrasts with host-cytoplasm/nucleus export reported in P. berghei; species difference unresolved\", \"Whether PTEX dependence reflects direct export of LISP2 or indirect developmental effect unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The biochemical activity of the LISP2 protein and its molecular targets in the parasite or host cell remain unknown, leaving open how LISP2 drives liver-stage completion and what it does once imported into the hepatocyte nucleus.\",\n      \"evidence\": \"No discovery in the corpus assigns a catalytic activity, binding partner, or nuclear target to LISP2\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct protein interaction partner identified\", \"No enzymatic or structural characterization\", \"Function of nuclear-imported LISP2 in host hepatocytes undefined\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008139\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020020\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0042025\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005737\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006606\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"importin-\\u03b1/\\u03b2 (mediates LISP2 nuclear import in P. falciparum) [#7]\",\n      \"EXP2 / PTEX150 (PTEX translocon; LISP2 expression/accumulation depends on them) [#6, #8]\",\n      \"PlasMei2 (synthetic-lethal genetic interaction in P. yoelii) [#2]\",\n      \"UIS3 (genetic interaction; combined deletion gives complete attenuation in P. berghei) [#3]\"\n    ],\n    \"other_free_text\": [\n      \"LISP2 is expressed in developing liver-stage schizonts but not in dormant hypnozoites, serving as a marker of active schizogony [#1, #5].\",\n      \"The LISP2 promoter is an autonomous liver-stage/schizont-specific regulatory element usable to drive stage-restricted reporter expression [#4, #5].\",\n      \"Genetic loss-of-function causes late liver-stage developmental arrest during merozoite formation across multiple Plasmodium species [#0, #2, #3].\",\n      \"Species divergence: exported to host hepatocyte cytoplasm and nucleus in P. berghei [#0], but retained at the parasite periphery (not exported beyond the PVM) in P. falciparum liver stages [#8].\",\n      \"The C-terminal bipartite NLS is phylogenetically unique to P. falciparum and P. reichenowi [#7].\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0405300","name":null,"product":"liver specific protein 2, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1003000","name":null,"product":"liver specific protein 2","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0303400","name":null,"product":"liver specific protein 2, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"LISP2","evaluation":null}