Identification And Functional Studies Of Eimeria Tenella Rhoptry Protein EtROP30 And EtROP35

Coccidiosis is one of the most harmful diseases that threaten the health and welfare of poultry,and brings huge economic losses to the poultry industry all over the world.However,due to technical difficulties such as in vitro culture of coccidia and gene manipulation,it is difficult to study the pathogenic mechanism,and the prevention and control of coccidiosis is progressing slowly.Through the previous analysis,we found that the rhoptry proteins（ROPs）might be virulence factor and play key roles in the interaction between the coccidia and the host,but studies on the ROPs of coccidia are limited.Among all the predicted E.tenella ROPs,EtROP30 is the only ROP containing both secretory signal peptide and nuclear localization sequence,implying that it may be secretory protein and enter host nucleus to regulate host cells.EtROP35 shows the highest transcriptional level among all E.tenella ROPs in the transcriptome analysis of sporozoite and merozoite,suggesting that it may have important functions.In this study,we identified and studied the function of EtROP30 and EtROP35,and explored function mechanism on the point of interaction between proteins and host cells.1.Identification and functions of EtROP30The EtROP30 gene is 1578 bp in length,encoding 525 aa,with a theoretical molecular weight of 56.69 KD.Sequence analysis suggests EtROP30 contains an 18 aa N-terminal secretion signal peptide,a protein kinase domain（175～510 aa）and a C-terminal nuclear localization signal sequence（516～525 aa）.EtROP30 is the only ROP containing both secretory signal and NLS in E.tenella.It contains eight E.tenella-specific rhoptry kinase 1subfamily（ROPK-Eten1）motifs（LGAGGTAV,EFAVKM,TER,CHADIKPENF,ADFGM,MDPSH,YDAWS,KRPTP）and 3 key residues(K²¹¹,D³⁶⁰,D³⁷⁸)required for protein kinase catalysis.The locations of EtROP30 are consistent with the location of the rhoptry protein.The secretion verification test of sporozoites showed that EtROP30 is a secreted protein.The sequence characteristics,localization and secretory characteristics of EtROP30 indicate that it belongs to rhoptry protein.Laser confocal observation and nucleocytoplasmic separation and Western blot detection showed that EtROP30 transferred to the nucleus in DF-1 cells,and the entering nucleus was mediated by the nuclear localization signal sequence.The relative levels of transcription and protein expression of EtROP30 in unsporulated oocysts,sporulated oocysts,sporozoites,and merozoites showed similar trends,and the expression levels in sporozoites and merozoites were significantly higher than those in unsporulated oocysts and sporulated oocysts,which is highest in the merozoite stage.Adhesion and invasion blocking tests showed that EtROP30 possessed the function of adhering to host cells in vitro and did not participate in the process of sporozoite invasion.In order to explore the significance of EtROP30 on the virulence of parasites,the T.gondii RHΔKu80-EtROP30 expressing EtROP30 was constructed by CRISPR/Cas9 system.It was found that the protein significantly promoted the intracellular proliferation of T.gondii and significantly enhanced its pathogenicity to mice.T.gondii is the model organism of apicomplexan parasites,so it is speculated that EtROP30 is the virulence factor of E.tenella.The expression of c FOS gene essential to the intracellular development of parasites was significantly up-regulated by RHΔKu80-EtROP30 compared with RHΔKu80.EtROP30enhanced the virulence of parasites may be due to the regulation of cellular c FOS.2.Screening,verification and functions of host cell proteins interaction with EtROP30In order to explore the regulation of EtROP30 on cells and the interaction with the host,this study carried out experiments on transcriptomics analysis of EtROP30 transfected cells,screening and verification of interaction proteins.Transcriptomics detected 927 differentially expressed genes,292 genes were up-regulated and 635 were down-regulated,including significantly up-regulated genes such as c FOS,EGR1 and CTGF,which are related to parasite infections,and the expression of type I interferon was significantly down-regulated.GO functional enrichment analysis showed that the differential genes were mainly related to STAT protein threonine phosphorylation,type I interferon-related signal pathways and related responses,immune response pathways,and cell receptor regulation.In order to screen the proteins that EtROP30 interacts with the host,immunoprecipitation was performed for protein gel silver staining,and the differential bands were screened and identified by mass spectrometry.The two differential bands identified 169 and 167 proteins respectively.Considering transcriptomics results and protein functions,22 proteins were further selected for verification by yeast two-hybrid test.It was found that EtROP30 did not interact with 18 of them,but interacted with 4 proteins（P53,STK26,TRIM68,and HSPA14）.These four proteins were verified by the intracellular co-localization and immunoprecipitation.The interaction between EtROP30 and STK26 was excluded.The remaining three proteins were verified by GST pull-down.GST pull-down reflects the direct interaction relationship between the proteins.The results revealed EtROP30 did not interact with P53 directly,but showed direct protein interaction with TRIM68 and HSPA14.In order to study the interaction region of EtROP30 with TRIM68 and HSPA14,EtROP30 was divided into three segments:19～174 aa,175～510 aa（kinase domain）,and 511～525 aa,and the yeast two-hybrid test was performed with TRIM68 and HSPA14,respectively.The results showed that none of the three segments interacted with TRIM68,which might have destroyed its interaction area by segmentation,or could not form the structure required for interaction after segmentation.The interaction region between EtROP30 and HSPA14 was the 175～510 aa.Whether the interaction related to kinase activity remained to be further studied.The overexpression of EtROP30 after si RNA interference with HSPA14 showed that apoptosis was induced by EtROP30 transfection,and the effect was down-regulated after down-regulation of HSPA14,which indicated that the mechanism of apoptosis induced by EtROP30 was through interaction with intracellular HSPA14.The specific pathway of this mechanism and whether there were other ways need to be further studied.3.Identification and functions of EtROP35The EtROP35 gene is 1467 bp in length,encoding 488 aa,with a theoretical molecular weight of 52.8 KD.Sequence analysis suggests EtROP35 contains an 25 aa N-terminal secretion signal peptide,a protein kinase domain（197～460 aa）containing eight ROP35subfamily motifs（KPRARLaa,GIVIKG,KEV,VHRDIKGCNY,ADFEG,IAPEL,TDVYA,NRYDI）and 3 key residues(K²⁵³,D³⁴⁴,D³⁶²)required for protein kinase catalysis.The locations of EtROP35 are consistent with the location of the rhoptry protein.The secretion verification test of sporozoites showed that EtROP35 is a secreted protein.The sequence characteristics,localization and secretory characteristics of EtROP35 indicate that it belongs to rhoptry protein.The relative levels of transcription levels of EtROP35 in sporulated oocysts,sporozoites,and merozoites showed significantly higher than unsporulated oocysts,which is highest in the merozoites stage.Adhesion and invasion blocking tests showed that EtROP35possessed the function of adhering to host cells in vitro and participated in the process of sporozoite invasion.The interacting proteins between EtROP35 and host cells were screened by immunoprecipitation combined with mass spectrometry,and their functions needed to be further studied.4.Evaluation of immune protection of EtROP30 and EtROP35In this study,the antigenicity of EtROP30 and EtROP35 and the immune protective effects of recombinant protein vaccines were evaluated.It was found that EtROP30 and EtROP35were natural antigens of coccidia and could induce to produce high levels of Ig Y.As recombinant protein vaccines,the immune protection evaluation tests of EtROP30 and EtROP35 showed that the average weight gain rate of the immune challenge groups were75.74%and 85.20%,which were significantly higher than that of the control group（52.33%）.The average lesion scores were 1.66±0.14 and 1.56±0.21,which were significantly lower than that of the control group（2.48±0.21）.The oocyst excretion decreased by 61.42%and 52.81%,and the anticoccidial index was 149.32 and 159.6.In summary,this study identified EtROP30 and EtROP35 and explored their functions,which indicated that they were ROPs of E.tenella.EtROP30 was the a virulence factor and EtROP35 played a certain role in adhesion and invasion.The regulation of EtROP30 on cells was analyzed by transcriptome,and the host proteins interacting with EtROP30 were screened by coimmunoprecipitation and mass spectrometry and verified by various methods.It was also found that the interaction between EtROP30 and HSPA14 regulated apoptosis.EtROP30 and EtROP35 were natural antigens of coccidiosis,and the recombinant protein vaccine could induce moderate anti-coccidiosis effects.This study provides a reference for screening the key pathogenic factors of coccidia and analyzing the pathogenic mechanism and prevention and control of coccidiosis.