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

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.

TgROP18 targets IL20RB for host-defense-related-STAT3 activation during Toxoplasma gondii infection

“…In our study, we were surprised to observe the activation of STAT3 in human keratinocyte (HaCaT) cells infected by the rop16 knockout RH strain (RH-∆ rop16 ). Combined with the finding of Tg ROP18-human IL20RB interaction in our previous research [ 35 ], we hypothesized that Tg ROP18 may activate STAT3 through binding with IL-20R to activate the JAK/STAT3 pathway. The HaCaT cell is a type of epithelial cell expressing both type I and type II IL-20 receptors in high levels, therefore, it was used as a cell model in this research.…”

“…In our previous study, a bi-molecular fluorescence complementation (BiFc) protein interaction screening was performed to identify the human interactom of Tg ROP18, and the human IL20RB was screened as an interaction protein of Tg ROP18 [ 35 ]. Fluorescence resonance energy transfer (FRET) was conducted to further confirm this interaction.…”

Toxoplasma gondiiROP18 Inhibits Human Glioblastoma Cell Apoptosis through Mitochondrial Pathway by Targeting Host Cell P2X1

“…To understand the molecular mechanism of Tg ROP18 inhibiting cell apoptosis and find its targets in host cells, we applied a genome wide screening of human targets for Tg ROP18 of RH strain with a Bi-molecular fluorescence complementation (BiFc) technique [25]. P2X1 was identified as a putative interacting partner of Tg ROP18.…”

TgROP18 Targets IL20RB for Host-Defense-Related-STAT3 Activation During T. gondii Infection

“…In our study, we were surprised to observe the activation of STAT3 in human keratinocyte (HaCaT) cells infected by the rop16 knockout RH strain(RH-∆rop16). Combined with the nding of TgROP18-human IL20RB interaction in our previous research (35), we hypothesized that TgROP18 may activate STAT3 through binding with IL-20R to activate the JAK/STAT3 pathway. The HaCaT cell is a type of epithelial cell expressing both type I and type II IL-20 receptors in high levels, therefore, it was used as a cell model in this research.…”