| Avian coccidiosis caused by one or more species of Eimeria is the most serious parasitic disease in poultry industry,resulting in$3 billion economic losses per year worldwide.Fully understanding the mechanism of coccidium invasion,parasitism,and pathogenic is the key to control avian coccidiosis,in which invasion into host cells is the first step to establish the infections of coccidiosis,and is also the best period to control avian coccidiosis.However,the mechanism study of coccidium invasion and their interaction with host cells is too much depending on that of toxoplasma and plasmodium,and also is limited in single proteins or factors,lacking of systematicness and pertinence.The bottleneck of the mechanism study is due to that chicken coccidium can not be effectively cultured in vitro.Also,the condition of cell culture is lack of standard,and there is an irregularity of invasion and development in the cells for coccidium.However,cell culture is still the most effective platform for studying the coccidium invasion mechanism and its interaction with host cells,currently.The present study aimed to systematacially screen the key genes or key proteins of host cells and chicken coccidium in the invasion process.E.tenella and DF1 cells were chosen as pathogeny and host cells,respectively,and the model of E.tenella invasion to host cells was established.The genes of DF1 cells in the invasion model were analyzed using RNA-seq technology,and proteins of both DF1 cells and E.tenella sporozoite in the invasion model were studied using i TRAQ technology,respectively.Two up-regulated uncharacterized candidate key proteins of E.tenella were selected for their functional study.The study provides data reference for the future study of mechanism of coccidium invasion and their interaction with host cells.The contents of this study include the following 5 parts:1.The establishment of E.tenella invasion to DF1 cells model in vitroIn order to establish the model of invasion to DF1 cells by E.tenella sporozoite in vitro,the invasion and development of E.tenella sporozoite in DF1 cells was studied,and the influence of E.tenella sporozoite dose and inoculation time to invasion rate of DF1 cells was investigated.The results indicated that the sporoziote successfully invaded into DF1 cells,and complished their development of the first generation of schizont and merozoite in the cells.The invasion rates of the same dose of sporozoite with different inoculation times and different doses with the same inoculation time were studied,respectively.The results revealed that the invasion rate increased with time,and there was a fast increase within 2 h,a slow after 2 h,and the invasion rate at 2 h was 33.14%.The dose of sporozoite could significantly influence its invasion rate:there was a positive correlation between the dose of sporozoite and invasion rate when the dose was less than 5×10~5,a negative one when the dose was higher than 5×10~5,and the invasion rate was 35.7%at the dose of 5×10~5.These results indicated that the model of E.tenella invasion to DF1 cells was successfully established,in which 2 h is a proper inoculation timepiont,with 5×10~5sporozoites inoculated to 2.5×10~5cells an appropriate inoculation density.2.Transcriptomics analysis of DF1 cells in E.tenella invasion modelTo integrally analyze the key genes of host cells in the process of E.tenella invasion,the comparative transcriptomics of DF1 cells in the invasion model was analyed using RNA-seq technology,with normal DF1 cells as control.A total of 5744 genes were identified in the infected DF1 cells,and 35 genes had significant changes,in which 32 genes were up-regulated,3 genes down-regulated.In the bioinformatics analysis,these significantly changed genes were all located in nucleus and transcription factor complex,with functions of transcription factor activity and sequence-specific DNA binding etc,participating in the regluration of RNA,nucleobase-containin compound biosynthetic process,and transcription from RNA polymerase II promoter,and were highly enriched in the signal pathway of MAPK.The results of real-time fluorescent quantitative PCR(qPCR)revealed a high coincidence rate between qPCR and transcriptomic results was up to 87.5%(7/8).Three genes(Fos B,NR4A3,and NR4A1)were both significantly up-regulated in both DF1 and CEF cells infected 2 h by sporozoites(P<0.01),and they were always highly transcribed in DF1 cells infected at 2 h,4h,and 8 h(P<0.05),with the highest level at 4 h.These results indicated that 35 significantly changed genes screened may could be the candidate key genes of host cells in the E.tenella invasion process,in which Fos B,NR4A3,and NR4A1 were important candidate key genes.3.Proteomics analysis of DF1 cells in E.tenella invasion modelIn order to analyze the key proteins of host cells in the E.tenella invasion process,the comparative proteomics of DF1 cells in the invasion model was analyzed using iTRAQ technology,with normal DF1 cells as negative control.A total of 3291 proteins were identified in the infected DF1 cells,and the number of significantly changed proteins was 302,in which 110 proteins were up-regulated,192 down-regulated.In the analysis of bioinformatics,these remarkably changed proteins were mainly located in nucleus,cytoplasm and mitochondria,with functions of nucleic acid binding,expression of genes,and the process and metablishm of RNA;the domain enrichment of these proteins was in the recognization of RNA and the binding of nucleic acid.The up-regulated proteins were highly enriched in Aminoacyl-t RNAbiosynthesis,Endocytosis,andPhagosomesignalpathways;down-regulated proteins were primarily enriched in Spliceosome signal pathway.4.Proteomics analysis of E.tenella sporozoite in the invasion modelTo analyze the key proteins of E.tenella during its invasion to host cells,the comparative proteomics of E.tenella sporozoite in the invasion model was analyzed using iTRAQ technology,with normal sporozoite as negative control.A total of 841 proteins were identified in E.tenella,and the number of significantly changed proteins was 81,in which 40 proteins were up-regulated,41 down-regulated.In the analysis of bioinformatics,these remarkably changed proteins primarily were organelle and supramolecular compounds composed of small ribosome subgroups,located in cytoplasm,nucleus,membrane and extracellular,with functions of binding and catalyze;the domain enrichment was in PH domain-like and Pyridoxal phosphate-dependent transferase(major region,subdomain 2).5.The functional study of E.tenella proteins U6KIV9 and H9B954Two up-regulated uncharacterized secretion proteins of E.tenella screened through proteomic,U6KIV9 and H9B954,were selected for their primary functional studies,including bioinformatic analysis,gene cloning and protein expression,preparation of polyclone antibodies,the role of adhesion and invasion to host cells,and the immunoprotection effects to chickens.The bioinformatics analysis results indicated that there were both signal peptide and transmembrane domain in the protein U6KIV9,without conserved domain;there was a signal peptide and MAR_sialic_bdg conserved domain in the protein H9B954.The genes of these two proteins were successfully cloned,and the proteins were expressed by E.coli and displayed on the surface of EBY100,and their polyclone antibodies were prepared using mice.The model of yeast surface displaying protein adhesion was established to study the adhesive effects of these two proteins to host cells,the results indicated that there was a significant cell adhesion effect for these two proteins,with the number of EBY100 displaying these two proteins adhered on DF1 cells significantly higher than those of empty plasmid displaying(P<0.05),also a higher adhesion rate of H9B954protein than U6KIV9(P>0.05).Polyclone antibodies were used to block these two proteins,and their invasion role to host cells were studied.The antibody blocking assay revealed that there was an obvious invasion effect for these two proteins,with a significant decrease of invasion rate to DF1 cells than normal mice serum(P<0.05),and the inhibition rate of U6KIV9 and H9B954 protein were up to 33.0±3.13 and 31.0±4.52,respectively.Immunized with these two proteins,there were significantly improved body weight gains(P<0.05),alleviated cecal lesions(P>0.05)and declined oocyst shedding(P<0.05)in the chickens compared with unimmunized individuals.These results indicated that both of E.tenella proteins U6KIV9,and H9B954,had invasion role,adhersion effect to host cells,and partial immunogenicity to animals. |
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