Formation of mammalian ER membrane contact sites, via host VAPA and VAPB, with the Toxoplasma PV

The Toxoplasma parasitophorous vacuole membrane (PVM) forms a barrier protecting the parasite from cellular assaults and serves as a platform for nutrient retrieval from the host cell. The PVM physically associates with many host organelles (e.g., ER and mitochondria), at distances reminiscent of membrane contact sites (MCS < 15 nm). We hypothesize that the parasite establishes functional MCS between the PVM and selected host organelles. In support, our RNAseq data show altered expression of several host lipid transfer proteins located at MCS (e.g., StAR, OSBP2, NPC1) in Toxoplasma-infected cells, suggesting hijacking of nonvesicular lipid transport pathways. Our fluorescence microscopy observations illustrate the recruitment at the PV of the host ER proteins VAPA and VAPB that both function as mammalian MCS tethering factors that bind to proteins with FFAT motifs. Our EM data show no host ER at PV in VAPA- and VAPB-deficient HeLa cells (DKO VAP, gift from P. De Camilli) infected up to 6h, suggesting Toxoplasma recognition of VAPA/VAPB for ER recruitment.However, 24h p.i. in DKO VAP cells, host ER was observed at the PV (27% coverage) though less than in WT HeLa (43%), suggesting that VAPA/VAPB are not critical for recruitment later in infection. We identified a PVM-localized Toxoplasma protein (we named TgVIP) with two putative FFAT motifs, as a potential host VAP interactor; a knock-out vip mutant exhibits a growth delay. Our EM data show TgVIP KO still recruits host ER, suggesting TgVIP is not involved in host ER-PVM attachment, but it may bind to host VAPs following host ER attachment to the PVM; we are assessing TgVIP and VAP interaction in situ. Previously, we demonstrated that Toxoplasma retrieves nutrients from host organelles (e.g., endolysosomes, lipid droplets, Rab vesicles) trapped in tubules of the intravacuolar network (IVN). The IVN-deficient parasite (∆gra2∆gra6), impaired in intra-PV host organelle sequestration, forms an extensive network of PVM projections (PVMP) extending into the host cell. As this mutant retains virulence, we hypothesize that its survival correlates with increased PVM-host organelle MCS formation. Indeed, host VAPA and a related host ER MCS protein MOSPD2 localize to ∆gra2∆gra6 PVMP. In DKO VAP cells at 24h pi, less host ER is recruited to ∆gra2∆gra6 PV and PV-associated host mitochondria show aberrant morphology. These observations point to a physiological relevance for MCS formation to support parasite infectivity.