Characterization of Membrane Contact Sites for the Facilitation of Lipid Exchange at the Malaria Parasite - Red Blood Cell Interface

Abstract

The malaria parasite maintains lipid homeostasis inside its host red blood cell. To that end, the parasite installs a lipid transport system with an unknown mechanism crossing two aqueous spaces: the red cell cytoplasm and the parasitophorous vacuole. We hypothesized that lipid transport across the parasitophorous vacuole may be facilitated at membrane contact sites. Using thin section electron microscopy, we measured the distance between the parasite plasma membrane (PPM) and the parasitophorous vacuole membrane (PVM). We found that two regions with distinct distances can be defined: 1) “PV lumenal” regions of 20-30 nm membrane distance and 2) regions of membrane apposition with ∼10 nm distance. To have a PV-lumenal label for live-cell microscopy we genetically targeted the fluorophore mRuby3 to the PV (PV-mRuby3). Correct targeting was confirmed using correlative light electron microscopy verifying that the discontinuous PV-mRuby3 signal shows the PV lumenal regions. To look for functions performed in each region, we localized two fluorescently tagged proteins relative to the PV-mRuby3: 1) EXP2, a channel involved in the export of proteins and import of nutrients across the PVM and 2) PfNCR1, a Niemann-Pick homologous lipid transporter in the PPM. Using correlation analysis on 2D projections of the parasite periphery, we found that the presence of EXP2 correlates well with the presence of PV lumen, while PfNCR1 is associated to regions of membrane apposition. Together both proteins complement to outline the periphery of the parasite. We conclude that membrane distance correlates with function of the host-parasite interface. The PV-lumen is a compartment that allows protein processing and storage. Lipid transport at sites of membrane apposition suggests a membrane contact site mediated mechanism for lipid exchange.