Screening Of Toxoplasma Gondii ROP32-interacting Proteins In Host Cells And Construction Of ROP32 Knockout Parasites

Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver a large amount of protein effectors into the host cell during parasite invasion. In order to achieve success intracellular invasion and proliferation in host cell, these effectors block the internal defense mechaneries of host cells by interacting with cellular factors and regulating of membrane and skeleton structures. T. gondii rhoptry proteins(ROPs) are indispensable secreted proteins involved in virulence, modifications of parasitophorous vacuole(PVM), modulations of host signalling and interactions between T. gonddi and host cells during intracellular replication and infection. Among the ROPs secreted into host cells by rhoptries, a T. gondii-specofic kinase family called ROPK may participate in a wide range of interactions with host cell proteins. Rhoptry protein 32(ROP32) is one of such ROPKs predicted to be active. Bioinformatic research found that most specific sites of ROPs are conserved, ROP32 in the only one kinase with a histidine at 358, while the aimino acid at the same site is conserved as a proline instead in other ROPKs. This phenominone may indicate that ROP32 assume the responsibility of specific biological functions.Currently, the specific functions of ROP32 are not clear. In this study, a partial sequence of ROP32 was successfully expressed and purified as a recombinant protein in E. coli. A partial sequence of ROP32 was cloned in the plasmid of pGBKT7 and used as bait vector to transform the yeast strain Y2 H. ROP32-interacting proteins were screened from a cDNA library constructed with HFF cells through a phenotypic screening method. The results showed that the bait plasmid pGBKT7-ROP32 has no self-activation and cytotoxicity, and 12 proteins interacted with ROP32 obviously were obtained all together. Sequence analysis indicated that positive clones comprised of a gene functioning in protein transportion, an apoptosis-related gene, a gene ecoding a cytoskeletal protein, a sequence encoding a phosphotransferase involved in energy conversion and other 8 sequences. In addition, the specific interaction between ROP32 and SEC63 was further validated using Co-IP. In order to investigate the function of ROP32 furtherly, we also constructed a ROP32 deficient parasite strain of T. gondii by using the CRISPR/Cas9 system. The growth and replication of ROP32 knockout parasites was evaluated in this study..In summary, a recombinant protein encoded by ROP32 were successfully expressed in this study. Twelvle host proteins were found to interact with ROP32 in the yeast two-hybrid assay. The interaction between ROP32 and SEC63 might suggest that ROP32 plays a role in protein transport into PVM of T. gondii. In addition, the ROP32 knockout strain, constructed in this study laid a foundation for further investigation of its functions.