Screening And Identification Of Interactors For Toxoplasma Gondii MIC6/7/8 And GRA15 And The Effects Of GRA15 On Host Cell Signal Transduction

Toxoplasma gondii is a zoonotic pathogen with a worldwide distribution,and is capable of infecting all warm-blooded animals.Microneme proteins(MICs),rhoptry proteins(ROPs)and dense granule antigens(GRAs)play important roles in invasion of Toxoplasma and survival of host cells during T.gondii infection.In the present study,the host proteins interacting with MIC6,MIC7,MIC8 and GRA15 proteins were screened and identified by yeast two-hybrid system.Also,the effects of each GRA15 of T.gondii PRU(Type II)and GT1(Type I)strains on host cell signal transduction were studied using RNA-seq technique and bioinformatics analysis.Yeast two-hybrid technique was used to verify the protein-protein interaction,using MIC6,MIC7 and MIC8 as prey proteins,and Spata3 and Dkk2 as bait proteins,respectively.The results showed that the MIC7 and MIC8 were able to interact with the host Spata3 and Dkk2 proteins,respectively,while the MIC6 protein was not.Given that Spata3 is a testis-specific protein,and Dkk2 plays an important role in the development of embryos,MIC7 and MIC8 may be involved in regulation of fetal development and host reproductive system.Using GRA15 of PRU strain as bait to screen a library expressing mouse proteins,we identified two host proteins,AW209491 and Luzp1,which can interact with GRA15.The Luzp1 protein harbors three nuclear localizing signals and is involved in regulating a subset of host non-coding RNA genes.Given that Luzp1 interacts with GRA15,we speculate that GRA15 may target the host cell nucleus or interfere with the function of Luzp1 by interacting with Luzp1.It was found that the GRA15 protein of GT1 and PRU strains had both common and unique modulation of the host cells by analyzing the transcripts of cells expressing GRA15 protein of GT1 and PRU strains.The GRA15 protein of PRU strain was mainly involved in the regulation of TNF,NF-?B,HTLV-I and NOD-like receptor signaling pathway.The GRA15 protein of the GT1 strain may assist the parasite in obtaining the fatty acid from the host cells.In summary,the host proteins interacting with MIC6,MIC7,MIC8 and GRA15 were screened and identified,and the effects of GRA15 proteins of GT1 and PRU strains on the host signaling pathways were investigated,for the first time.The results not only deepen ourunderstanding of molecular pathogenesis of T.gondii infection,but also provide the theoretical basis for the development of new drugs and vaccines against T.gondii infection.