Genome-Wide Bimolecular Fluorescence Complementation-Based Proteomic Analysis of Toxoplasma gondii ROP18’s Human Interactome Shows Its Key Role in Regulation of Cell Immunity and Apoptosis

Abstract

<i>Toxoplasma gondii</i> rhoptry protein ROP18 (<i>Tg</i>ROP18) is a key virulence factor secreted into the host cell during invasion, where it modulates the host cell response by interacting with its host targets. However, only a few <i>Tg</i>ROP18 targets have been identified. In this study, we applied a high-throughput protein-protein interaction (PPI) screening in human cells using bimolecular fluorescence complementation (BiFC) to identify the targets of Type I strain ROP18 (ROP18_I) and Type II strain ROP18 (ROP18_II). From a pool of more than 18,000 human proteins, 492 and 141 proteins were identified as the targets of ROP18_I and ROP18_II, respectively. Gene ontology, search tool for the retrieval of interacting genes/proteins PPI network, and Ingenuity pathway analyses revealed that the majority of these proteins were associated with immune response and apoptosis. This indicates a key role of <i>Tg</i>ROP18 in manipulating host's immunity and cell apoptosis, which might contribute to the immune escape and successful parasitism of the parasite. Among the proteins identified, the immunity-related proteins N-myc and STAT interactor, IL20RB, IL21, ubiquitin C, and vimentin and the apoptosis-related protein P2RX1 were further verified as ROP18_I targets by sensitized emission-fluorescence resonance energy transfer (SE-FRET) and co-immunoprecipitation. Our study substantially contributes to the current limited knowledge on human targets of <i>Tg</i>ROP18 and provides a novel tool to investigate the function of parasite effectors in human cells.