Screening and Identification of the Host Proteins Interacting with Toxoplasma gondii Rhoptry Protein ROP16

Abstract

Toxoplasma gondii, as a zoonotic protozoan parasite, develops sophisticated strategies to manipulate hosts for efficient intracellular survival. After successful invasion, T. gondii injects many effector proteins into host cells for various purposes. TgROP16 (T. gondii rhoptry protein 16), which is secreted from rhoptries into host cells, can activate the host STAT (signal transducer and activator of transcription) signaling pathway through phosphorylation of STAT3 and STAT6. However, whether there are other host proteins modulated by TgROP16 is currently unknown. In this study, yeast two-hybrid (Y2H) screen was used to look for additional host proteins interacting with TgROP16. Yeast cells expressing a mouse cDNA library cloned into the prey vector were used to mate with yeasts expressing ROP16 without signal peptide. Two mouse proteins, Dnaja1 (DnaJ heat shock protein family member A1) and Gabra4 (gamma-aminobutyric acid A receptor, subunit alpha 4) were identified to interact with ROP16 from this screen. Further analysis suggested that the Predomain of ROP16 played key roles in mediating interactions with these host proteins, whereas the contribution from the Kinase domain was minor. The interactions between Dnaja1 and different parts of ROP16 were also estimated in vivo by co-immunoprecipitation. The results showed that the Predomain of ROP16 was the major region to interact with Dnaja1, which is consistent with the Y2H results. Based on the gene ontology analysis, Dnaja1 is predicted to participate in stress response while Gabra4 is involved in the system development process. The discovery of new host proteins that interact with ROP16 of T. gondii will help us to further investigate the functions of this effector proteins during T. gondii infection.

Genome-Wide Identification of the TIFY Family in Salvia miltiorrhiza Reveals That SmJAZ3 Interacts With SmWD40-170, a Relevant Protein That Modulates Secondary Metabolism and Development

“…The autoactivation test of the SmJAZ3 in the yeast was conducted as previously described ( Li et al, 2018 ). Based on the previously described protocols ( Pan et al, 2017 ), the S. miltiorrhiza cDNA library expressed in the Y187 yeast cells was mated with the AH109 yeast cells expressing SmJAZ3, and then screened for the interacting partners of SmJAZ3.…”

Life cycle stages, specific organelles and invasion mechanisms ofEimeriaspecies

“…TgSORTLR loads MIC and ROP/RON proteins at the TGN and escorts them to the ELC (Sloves et al ., 2012; Sangare et al ., 2016). In higher eukaryotes, sortilin is a transmembrane endosomal receptor with a major role in anterograde transport of lysosomal enzymes from TGN to endosomes (Bonifacino and Rojas, 2006), and in retrograde trafficking where it binds retromer, an evolutionarily conserved protein complex that selects and recycles proteins from endosomes to the TGN or the plasma membrane (Pan et al ., 2017). Recent characterization of retromer interactomes in T. gondii confirms that retromer-dependent retrograde transport is essential for apical organelle biogenesis, probably because recycling of TgSORTLR to the TGN is needed to maintain anterograde trafficking of MIC and ROP/RON proteins to the ELC (Sangare et al ., 2016).…”

Comparison of splenocyte microRNA expression profiles of pigs during acute and chronic toxoplasmosis

“…T. gondii develops sophisticated strategies to manipulate hosts for efficient intracellular survival [15]. A recent study showed that different T. gondii strains differed in their ability to induce host immune responses and signaling pathways [16].…”