Abstract
Toxoplasma gondii rhoptry protein ROP18 (TgROP18) 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 TgROP18 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 (ROP18I) and Type II strain ROP18 (ROP18II). From a pool of more than 18,000 human proteins, 492 and 141 proteins were identified as the targets of ROP18I and ROP18II, 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 TgROP18 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 ROP18I 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 TgROP18 and provides a novel tool to investigate the function of parasite effectors in human cells.
Highlights
Toxoplasma gondii is an obligate intracellular protozoon that causes zoonotic toxoplasmosis
We found an interaction between ROP18I and Fas apoptotic inhibitory molecule (FAIM) in this study, which suggested that the parasite might interfere with the death receptor pathway of host cell apoptosis through targeting the key component in this pathway by Toxoplasma gondii rhoptry protein ROP18 (TgROP18)
We used high-throughput protein–protein interaction (PPI) screening based on bimolecular fluorescence complementation (BiFC) for the first time to identify the human host proteins targeted by the T. gondii key virulence factor TgROP18
Summary
Toxoplasma gondii is an obligate intracellular protozoon that causes zoonotic toxoplasmosis. BiFC Analysis of TgROP18 discharges a family of proteins termed rhoptry proteins (ROPs) that are of importance for host cell invasion, intracellular survival, and interference with host functions [3, 4]. T. gondii isolates collected from North America and Europe primarily fall into one of the three distinct clonal lineages, types I, II, and III [5], which present a number of different phenotypes, such as growth, migration, and transmigration [6]. TgROP18 is a serine/threonine kinase secreted from the rhoptry into the parasitophorous vacuole membrane (PVM) and host cytosol during parasite invasion [13], of which the Type I strain (ROP18I) (RH strain, GenBank accession NO: AFO54817.1) and the Type II strain (ROP18II) (ME49, GenBank accession NO: XP_002367757.1) are different at 28 amino acid sites
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