PIPLC

TgPI-PLC is a plasma membrane-localized phosphatidylinositol-specific phospholipase C of Toxoplasma gondii that couples lipid hydrolysis to intracellular Ca2+ signaling governing tachyzoite motility and egress (PMID:16288600, PMID:29946311). The cloned enzyme is a ~121 kDa polypeptide organized as an N-terminal PH domain, an EF-hand motif, the catalytic X and Y domains, and a C-terminal C2 domain, and both recombinant and native enzyme preferentially hydrolyze phosphatidylinositol over PIP2 (PMID:16288600). Functionally, PI-PLC acts together with PKG to control cytosolic Ca2+ signals mobilized from acidic organelles, linking the enzyme to MIC2 secretion, gliding motility, and egress (PMID:29946311). Independently, PI-PLC has served as a definitive biochemical probe establishing that the major tachyzoite surface antigens—P30/SAG1, P43, P35, P22, and gp23—are attached to the plasma membrane through GPI anchors, since PI-PLC cleavage renders them water-soluble, removes their fatty-acyl labels, and exposes a trypanosome-VSG-like cross-reacting determinant (PMID:2925621, PMID:2531282, PMID:8241958). Used as a topological tool in permeabilized parasites, PI-PLC also mapped early GPI biosynthetic intermediates to the cytoplasmic leaflet of the ER and revealed an inositol-acylated, PI-PLC-resistant intermediate (PMID:16896225).

1. 1989 High

   Establishing how major surface antigens attach to the parasite membrane was open; PI-PLC cleavage demonstrated that P30/SAG1 and the other major tachyzoite surface antigens are GPI-anchored via a diacylglycerol-containing glycolipid.

   Evidence Metabolic radiolabeling, PI-PLC treatment of intact parasites and lysates, PLA2 digestion, and PAGE mobility shift/immunoprecipitation in T. gondii tachyzoites

   PMID:2925621 PMID:2531282

   Open questions at the time

   • Did not identify the enzyme's endogenous parasite gene
   • GPI core glycan fine structure not fully resolved in these reports
2. 1993 High

   Extending the GPI-anchor model to additional surface glycoproteins, PI-PLC release of gp23 combined with glycan analysis defined a conserved GPI core bearing beta-linked N-acetylgalactosamine.

   Evidence Multi-radiolabel metabolic labeling, PI-PLC and PLA2 digestion, chemical dephosphorylation/deamination, and exo/endoglycosidase analysis in living T. gondii

   PMID:8241958

   Open questions at the time

   • Biological function of the GalNAc modification not addressed
   • Did not link cleavage to the endogenous PI-PLC enzyme
3. 2006 High

   The identity and biochemical properties of the parasite enzyme itself were unknown; cloning and recombinant expression defined a PH-EF-X/Y-C2 phospholipase C that preferentially hydrolyzes PI over PIP2 and localizes to the plasma membrane.

   Evidence Molecular cloning, recombinant expression in E. coli, in vitro PI/PIP2 hydrolysis assays, and indirect immunofluorescence in T. gondii

   PMID:16288600

   Open questions at the time

   • No genetic knockout to establish essentiality or in vivo function
   • Regulation of catalytic activity not characterized
4. 2006 Medium

   The membrane topology of GPI biosynthesis was unresolved; using PI-PLC as a leaflet-specific probe placed early GPI intermediates in the cytoplasmic ER leaflet and revealed a PI-PLC-resistant inositol-acylated intermediate.

   Evidence Hypotonic permeabilization, metabolic labeling of GPI intermediates, PI-PLC probing of transmembrane distribution, protease protection, and electron microscopy in T. gondii

   PMID:16896225

   Open questions at the time

   • Uses PI-PLC as a reagent rather than testing its own cellular role
   • Enzyme(s) performing inositol acylation not identified
5. 2018 Medium

   Whether PI-PLC contributes to Ca2+-driven invasion/egress behavior was untested; pharmacological inhibition placed PI-PLC with PKG upstream of cytosolic Ca2+ mobilization from acidic stores, MIC2 secretion, gliding, and egress.

   Evidence Pharmacological inhibition of PI-PLC and PKG with cytosolic Ca2+ flux measurement, MIC2 secretion, gliding and egress assays in T. gondii

   PMID:29946311

   Open questions at the time

   • Pathway placement rests on inhibitors, not genetic knockout
   • Direct enzymatic product coupling Ca2+ release not demonstrated
6. 2020 Low

   Whether PI-PLC influences secretory-antigen expression was addressed by inhibitor treatment, which reduced GRA1 and MIC3 mRNA and protein in vitro and in vivo.

   Evidence Edelfosine (PI-PLC inhibitor) treatment of infected Vero cells and a mouse model, qRT-PCR and Western blot in T. gondii

   PMID:31970471

   Open questions at the time

   • Pharmacological inhibition only, no genetic manipulation of PI-PLC
   • Edelfosine effects may be indirect on the enzyme

• The endogenous, genetically defined function of TgPI-PLC in Ca2+ signaling and its physiological lipid substrate/product in vivo remain to be established.
• No conditional knockout or complementation of TgPI-PLC reported
• In vivo substrate preference (PI vs PIP2) during signaling not resolved
• Mechanism linking PI-PLC activity to acidic-store Ca2+ release undefined