Abstract
Membrane contact sites (MCS) are essential for organelle communication in eukaryotic cells. Pathogens also establish MCS with host organelles, but the mechanisms underlying these interactions and their role in infection remain poorly understood. Here, using a fluorescence sensor and CRISPR-based loss-of-function screening, together with imaging and proteomics, we identify the parasite effector mediating MCS between host endoplasmic reticulum (ER) and the vacuole containing the intracellular parasite Toxoplasma gondii. TgROP1 acts as a tether and mimics a canonical FFAT motif to bind the host ER proteins VAPA and VAPB. The loss of VAPA/B abolished host ER-Toxoplasma MCS and decreased pathogen growth. These findings indicate that targeting of host MCS tethers is a strategy exploited by pathogens during infection, which could inform future treatment design.