PLP2

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).

1. 2013 High

   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

   PMID:23461714

   Open questions at the time

   • Did not resolve how PLP2 discharge is triggered or regulated
   • No structural or biochemical characterization of the pore in this study
2. 2013 Low

   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

   PMID:23591903

   Open questions at the time

   • Expression confirmation only; no functional data for PLP2 in this study
   • Mechanistic work concerned PLP1, not PLP2
3. 2014 High

   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

   PMID:24602217

   Open questions at the time

   • Molecular trigger linking calcium to discharge not defined
   • Pore architecture and lipid selectivity not determined
4. 2019 Medium

   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

   PMID:31413195

   Open questions at the time

   • Whether PfPATPL1 acts directly or through membrane remodeling is unresolved
   • Single-lab observation
5. 2020 Medium

   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

   PMID:31680162

   Open questions at the time

   • Method-validation context rather than new mechanism
   • Single lab
6. 2022 Medium

   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

   PMID:35598848

   Open questions at the time

   • Full-length pore assembly not resolved in this study
   • Single study
7. 2026 High

   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

   PMID:42675073

   Open questions at the time

   • 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

• The molecular event coupling calcium signaling and PfPATPL1 activity to the timing and site of PLP2 pore assembly on the erythrocyte membrane remains undefined.
• No mechanism linking calcium-triggered discharge to membrane-side pore nucleation
• No in situ structure of PLP2 pores on native gametocyte-infected erythrocyte membranes