LCAT

TgLCAT is a secreted phospholipase of Toxoplasma gondii that is stored in a subpopulation of dense granule organelles and, upon secretion, localizes to the parasitophorous vacuole and the parasite plasma membrane, where it acts on host- and parasite-derived membranes to promote tachyzoite egress (PMID:26694607). The enzyme carries a serine lipase motif ('AHSLG') and a Ser-Asp-His catalytic triad, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that each retain part of the triad and must reassemble to reconstitute activity (PMID:26694607). Catalytically, TgLCAT hydrolyzes phosphatidylcholine to generate membrane-disruptive lysophosphatidylcholine and transesterifies the released fatty acid onto cholesterol, though with lower transesterification activity than mammalian LCAT (PMID:26694607). This catalytic activity is required for its biological role: a catalytically inactive mutant fails to rescue the egress defect of LCAT-deficient parasites, and loss of LCAT delays or blocks efficient egress from host cells (PMID:29507893).

1. 2015 Medium

   Establishing where TgLCAT resides and what enzymatic machinery it carries defined it as a secreted dense-granule phospholipase acting at the host-parasite interface.

   Evidence Catalytic-motif sequence analysis plus organelle fractionation and localization imaging in T. gondii

   PMID:26694607

   Open questions at the time

   • Mechanism of dense-granule sorting and secretion timing not defined
   • Precise membrane targets at the PV versus plasma membrane not resolved
2. 2015 High

   Reconstituting activity from two proteolytic fragments revealed an unusual maturation mechanism distinguishing TgLCAT from canonical LCAT enzymes.

   Evidence In vitro enzymatic reconstitution assay from proteolytic fragments in T. gondii

   PMID:26694607

   Open questions at the time

   • Identity of the protease responsible for cleavage unknown
   • Structural basis of fragment reassembly not determined
3. 2015 High

   Defining the substrate and products showed TgLCAT generates membrane-disruptive lysophosphatidylcholine while transferring fatty acid to cholesterol, linking its biochemistry to membrane perturbation.

   Evidence In vitro lipid substrate assay measuring product formation in T. gondii

   PMID:26694607

   Open questions at the time

   • Physiological membrane substrate in vivo not identified
   • Functional significance of lower transesterification activity versus mammalian LCAT unclear
4. 2015 Medium

   Loss- and gain-of-function genetics tied TgLCAT to replication, virulence, and egress, positioning it as a facilitator of the intracellular lytic cycle.

   Evidence Genetic knockout and overexpression with growth, in vivo virulence, and egress assays in T. gondii

   PMID:26694607

   Open questions at the time

   • Whether growth/virulence defects are direct or secondary to egress defect not resolved in this study
   • Molecular events downstream of egress not defined
5. 2018 High

   Active-site mutagenesis with complementation established that TgLCAT's catalytic activity is specifically required for egress, isolating egress as its primary function.

   Evidence Genetic complementation with catalytically inactive mutant and egress assay across multiple independent LCAT-deficient lines in T. gondii

   PMID:29507893

   Open questions at the time

   • The absence of growth/virulence defects here versus the 2015 study not reconciled
   • Direct in situ demonstration of membrane lysis during egress lacking

• How TgLCAT-generated lysophosphatidylcholine drives membrane rupture during egress at the molecular level remains unresolved.
• No structural model of the reassembled active enzyme
• Protease and regulation of fragment maturation unknown
• In situ demonstration linking lipid products to egress membrane disruption absent