A high-throughput screen to elucidate the function of Toxoplasma gondii rhoptry proteins within human host cells

Toxoplasma gondii contain apicomplexan-specific rhoptry organelles whose proteins are essential in mediating invasion of host cells and host-pathogen interactions at early intracellular timepoints. Rhoptry proteins (ROPs) are deposited within the host prior to invasion and localize to various host compartments to rewire the host cell for the parasite"s survival. While some of these proteins have been characterized, the function of the majority of ROPs remains unknown. Here we systematically screen the function of ROPs within host cells utilizing a BioID approach, where ROP baits fused to miniTurboID, are expressed in HEK293 cells and biotinylated proximal prey are identified by mass spectrometry. We have generated a partial dataset containing 23 rhoptry baits and 1472 prey. These data recapitulate known features of rhoptry host biology. Toxofilin prey are significantly enriched in actin cytoskeleton localizing proteins, including the known interactor actin. ROP18 prey are significantly enriched in endoplasmic reticulum proteins, including the known substrate RPN1. Furthermore, ROPs that localize to host compartments do so in this experimental system: ROP47 and PP2C-hn prey are significantly enriched in nucleoplasm proteins and ROP39 prey are significantly enriched in mitochondrial proteins. Novel insights include an enrichment of microtubule proteins in ROP19A prey and peroxisomal proteins in ROP13 prey. Together these results establish the efficacy of this approach and highlight the potential for novel discoveries regarding the function of rhoptry proteins in human host cells.