Screening And Characteristics Of Differentially Expressed Proteins Of BHK-21 Cells Infected With Eimeria Tenella

Chicken coccidiosis occupies a high incidence in chickens in various epidemics.According to statistics,the mortality rate is as high as 30%,causing great economic losses to the breeding industry.But so far,there have been few reports of host cell proteins associated with the invasion of Eimeria.In order to study the proteins function of host cells involved in invasion,development and proliferation,we used isobaric tags for relative and absolute quantitation(iTRAQ)coupled with LC-MS/MS to analyze the differentially expressed proteins(DEPs)of BHK-21 cells infected with Eimeria tenella sporozoites 24 h post-infection(p.i.),and three DEPs,receptor for activated C kinase 1(RACK1),metastasis associated gene 3(MAT3)and lamin B receptor(LBR),were selected to further study their characteristics.The results lay the foundation for further investigation of the interaction between Eimeria and host.1.iTRAQ-based comparative proteomic analysis of BHK-21 cells infected with Eimeria tenella sporozoitesEimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens.When E.tenella infects a host cell,the host produces a corresponding change to deal with damage caused by this infection.To date,the mechanism of how the host cell responds to an E.tenella infection is highly limited at both the molecular and cellular levels.In this study,iTRAQ coupled with LC-MS/MS was used to screen DEPs in BHK-21 cells infected with E.tenella sporozoites for 24 h.p.h.In total,6139 non-redundant distinct proteins were identified and 195 of these were found to have a fold change ratio ? 1.3 or ? 0.7 and P < 0.05,including 151 up-regulated proteins and 44 down-regulated proteins.qRT-PCR and Western blot were used to verify the reliability of Mass spectrometry data.GO analysis indicated that the up-regulated DEPs were mainly related to binding and catalytic activity,whereas the down-regulated were catalytic activity and molecular function regulators.Furthermore,KEGG pathway analysis showed that the DEPs were involved in the PI3K-Akt signaling pathway,RIGI receptor-mediated phagocytosis pathway,Ras signaling pathway and the p53 signaling pathway.Additionally,it was found that the up-regulated and down-regulated DEPs were mainly associated with the ribosome and mRNA surveillance pathway,respectively.Collectively,the data processed in this investigation provides a useful basis for more detailed analyses the interaction between Eimeria and host.2.Effect of Eimeria tenella sporozoites infection on host's RACK1In the present study,one of those host's DEPs,RACK1,was cloned by PCR with the cDNA of chicken muscle's as template.The target segments were subcloned to pGEX-6P-1 vector and the prokaryotic recombinant expression plasmid p GEX-6P-1-RACK1 was constructed.The RACK1-GST was expressed and purified.The protein RACK1-GST was identified by western blot.The result of western blot showed that a protein about 61 kDa was recognized by anti-GST antibody confirming that the obtained protein was RACK1-GST fusion protein.The transcriptional level and translational level of RACK1 in DF-1 cells during Eimeria tenella sporozoites infection were detected by RT-qPCR and western blot.The results showed that both of transcriptional level and protein expression level of RACK1 in cells at 24 ~ 72 h post-infection were significantly increased.The effect of RACK1 polyclonal antibody on sporozoites invasion of cells was detected by the antibody inhibition assay.The results showed that antibodies of 100 ?g/mL and 200 ?g/mL had little influence on the sporozoites invasion ability,while antibodies of 300 ?g/mL and 400 ?g/mL can significantly increase sporozoites invasion ability.These results showed that host's RACK1 may play a negative role on Eimeria invasion.However,further studies are needed to illuminate the exact mechanism of how RACK1 negatively regulate Eimeria invasion.3.Effect of Eimeria tenella sporozoites infection on host?s MTA3 and LBRIn order to explore the role of LBR and MTA3 invade into host cells,we examined the changes in transcriptional levels and translational levels of LBR and MTA3 in cells infected with sporozoites at 24~72 h post-infection and the effect of LBR and MTA3 antibodies on sporozoites invade into cells.The results showed that with the increase of invasion time,the transcriptional and translational levels of MTA3 were increased continuously.The 24 h group was significantly higher than the non-infected group(P<0.05),while the protein expression levels of the 48 h and 72 h groups were significantly higher than the 24 h group and non-infected group(P<0.05).However,the transcriptional levels and translation levels of LBR were decreased with the increase of invasion time.The 24 h group was lower than the non-infected group,but the difference was not significant(P>0.05),while the protein expression levels of the 48 h and 72 h groups were significantly lower than the 24 h group and non-infection group(P <0.05).When the cells were treated with MTA3 or LBR antibodies of 10 ?g/mL and 20 ?g/mL,it had little influence on the sporozoites invasion ability.And the invasion rate of 10 ?g/mL and 20 ?g/mL was comparable to the control groups and normal IgG groups(P>0.05).The results may be related to the low concentration of antibody used(10-20 ?g/mL).Subsequently,invasion inhibition test of sporozoite are required to do with a higher concentration of antibodies,which can ensure whether MTA3 and LBR are associated with coccidia invasion.4.Effect of Eimeria tenella sporozoites infection on host cell apoptosisBy the KEGG analysis of DEPs,we predicted that some differentially expressed proteins involved in apoptosis-related pathways,such as MAPK,P53,Wnt,Ras and other apoptosis-related signaling pathways.In order to further investigate the effect of infection of Eimeria tenella sporozoites on host cell apoptosis,we examined apoptotic enzymes in the apoptotic pathway.Compared with the control group,the enzyme activities of Caspase-3 and Caspase-9 in cell infected with sporozoites were significantly increased at 12~24h post-infection(P<0.05)with the increase of invasion time.It showed that the invasion of sporozoites would accelerate the apoptosis of host cells,but the specific mechanism needs to further study.