Identification And Functional Analyses Of Secretory Dense Granule And Rhoptry Proteins In Toxoplasma Gondii

Toxoplasma gondii is a highly successful zoonotic pathogen belonging to apicomplexan parasites.As a unicellular parasitic protozoan,it can infect all warmblooded animals and all nucleated cells of these hosts.To survive in such diverse cell types and hosts,Toxoplasma gondii relies on a large number of secretory proteins to mediate interactions with the hosts and ensure the growth of parasites.Dense granule proteins and rhoptry proteins are two main classes of secretory proteins that play important roles in the growth and development of parasites as well as immune evasion process.But up to now,only a small number of GRA proteins have been identified and more are still to be discovered.On the other hand,a number of ROPs have been discovered,but their working mechanisms are not very clear.Therefore,in this study we used protein labeling techniques to identify novel GRA proteins and yeast twohybrid to look for host proteins that interact with Toxoplasma ROP proteins.The main research work include:?1?Identification and functional study of novel dense granule proteinsUsing the two proximity-based protein labeling approaches,APEX and BirA*,and the dense granule protein GRA1 as a bait,we biotinylated the potential GRA proteins of Toxoplasma gondii and finally successfully identified 46 proteins,including 20 known GRA proteins and 26 candidates.Of the 46 proteins,17 were identified by both APEX and BirA* based methods,and 14 out of 17 were known GRA proteins,while the other 3 were also confirmed to be located in dense granules in this study.Other GRA proteins were identified either APEX or BirA* based approach,indicating that these two methods are complementary.Using protein localization analyses,we identified these GRA candidates and five of them were verified as novel dense granule proteins.Subsequently TGGT1₂00360 and TGGT1₃19340 were subject to gene disruption studies,and the results showed that the deletion of the former did not affect parasite growth in vitro and in vivo,but the latter inactivation is probably lethal.Based on the homology alignment of these 26 GRA candidates in different apicomplexan parasites,we found that most of the proteins have high phenotype scores,and these proteins are only found in Toxoplasma and several other closely related parasites;while a small portion of proteins have a very low value,and these proteins are widely present in Cryptosporidum,Hematozoa etc.Therefore,we speculate that GRA proteins can be divided into two categories based on the functions: conserved ones with lower phenotype scores,which may be involved in parasite growth under all conditions;less conserved ones with higher phenotype scores such as TGGT1₂00360,which may help the parasites adapt to different host environments instead of the parasite growth in vitro.?2?Screening and identification of host proteins interacting with the rhoptry proteins ROP16In the yeast two-hybrid experiment with bait protein Toxoplasma gondii ROP16 and mouse brain cDNA library,we successfully obtained two positive clones interacting with ROP16,and confirmed two mouse proteins Dnaja1 and Gabra4 by sequencing and alignment.According to gene function analysis,ROP16 may participate in host immune response and regulation of the nervous system through these two proteins.Point-to point yeast two-hybrid verified the interaction between ROP16 and Dnaja1 or Gabra4.In addition,we also identified the predomain of ROP16 is key to mediate interactions with these two host proteins.The ROP16 gene and mouse gene Dnaja1/Gabra4 genes were cloned into eukaryotic vector respectively.After transfection into 293 T cells successfully,we confirmed the interaction between ROP16 and Dnaja1 by coimmunoprecipitation experiments.With the same method we also confirmed that the predomain of ROP16 plays a key role in mediating the interactions.In this study,proximity-based protein biotinylation was used to identify new dense granule proteins in Toxoplasma gondii and 26 candidates were discovered,among which 5 were confirmed to be novel GRA proteins.On the other hand,yeast two-hybrid was used to screen for host proteins interacting with ROP16 and two mice proteins were successfully identified.The identification of novel GRA proteins further deepened our understanding of secretory proteins in Toxoplasma gondii,and shed new lights on the research on parasite growth and host modulation.The study of host proteins interacting with ROP16 provided new basis towards the complete dissection of the function of ROP16.Taken together,this study focused on secretory proteins in Toxoplasma gondii,the identification of new components and functions facilitated further analysis of secretory proteins and paved the way for full understanding of host-parasite interactions.