Extensive membrane lipid remodeling at the host-parasite interface of Toxoplasma gondii

Phosphoinositides regulate diverse membrane trafficking and signaling events in eukaryotic cells, yet their transbilayer distribution remains largely unexplored, particularly at host-parasite membrane interfaces. Here, we determined the membrane leaflet localization of phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in Toxoplasma gondii using quick-freezing freeze-fracture replica labeling combined with phosphoinositide-specific probes. Both phosphoinositides were predominantly localized to the cytoplasmic leaflet of the parasite plasma membrane, whereas only weak labeling was detected in the inner membrane complex. PtdIns(4)P, but not PtdIns(4,5)P2, was additionally detected on intracellular vesicles, while mature micronemes lacked detectable labeling for either phosphoinositide. Surprisingly, both phosphoinositides exhibited nearly symmetric distributions between the two leaflets of the parasitophorous vacuole membrane (PVM), in striking contrast to their asymmetric localization in the parasite plasma membrane. Together with our previous demonstration that phosphatidylserine and phosphatidylethanolamine also lose their transbilayer asymmetry in the PVM, these findings indicate that the PVM undergoes global membrane lipid remodeling that extends from major phospholipids to phosphoinositides. We propose that this extensive lipid reorganization represents a defining structural feature of the apicomplexan PVM and provides a new framework for understanding membrane identity at the host-parasite interface.