Study On The Effect Of Ethanamizuril On The Second-generation Merozoites Proteome Of Eimeria Tenella

Coccidiosis is a parasitic intestinal disease caused by Eimeria,resulting in huge economic losses in the global poultry industry every year.Eimeria tenella(E.tenella)has a strong pathogenicity.In the second-generation merozoites,trophozoites undergo multiple fission to form schizonts,which further generates numerous merozoites,causing irreversible damage to intestine.So it is very important to study the effect of drugs on the second-generation of merozoites.The control of E.tenella mainly depends on prophylactic chemotherapy.Ethanamizuril is a novel triazine compound independently developed by Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics,Ministry of Agriculture and Rural Affairs,with the characteristics of broad spectrum,high efficiency and low toxicity.The previous studies showed that the anticoccidial activity of Ethanamizuril was most significant in the second-generation merozoites.In order to explore the molecule of action and the potential resistant mechanism of Ethanamizuril,we studied the effect of Ethanamizuril on the second-generation merozoites proteome of E.tenella.The results are as follows:1.Using the method of label free proteomics,the effect of Ethanamizuril on the protein expression level in the second-generation merozoites of E.tenella was studied,and the target proteins with drug effect were screened out.The quantitative analysis and identification of proteins were carried out by liquid chromatography-mass spectrometry/tandem mass spectrometry(LC-MS/MS).Then these proteins were enriched by the methods of Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and Protein-protein interaction networks(PPI),in which 145 proteins with significant difference in expression were screened out,including 66 up-regulated proteins and 79 down-regulated proteins.GO analysis showed that the up-regulated proteins involved in gene transcription,protein synthesis and metabolism,and the down-regulated proteins mainly included ribosome proteins and surface antigens(SAGs).KEGG pathway and PPI network suggested that differential proteins were mainly involved in RNA metabolism and protein biosynthesis.Interestingly,there were 17 SAGs,which accounted for the largest proportion in the significantly down-regulated proteins of E.tenella.It is speculated that SAGs are the most likely key molecule of drug action.2.In order to verify the above hypothesis,we selected ten SAGs to detect the change of the relative expression of SAGs in the mRNA level of the second-generation merozoites of E.tenella by Real-time PCR.The results showed that the mRNA level of SAGs in the sensitive strains treated with Ethanamizuril were down-regulated,but that of the resistant strains was reversed.3.In order to study the effect of Ethanamizuril on the protein levels of SAGs in the second-generation merozoites,two SAGs(SAGfm1 and SAGfm2)were selected,whose gene expression levels were significantly reversed.The expression of SAGs in vitro was realized by gene cloning and Escherichia coli(E.coli)expression system.The result of SDS-PAGE showed that the two recombinant proteins were inclusion body proteins both with specific fragments at 32 kD.Western blot analysis showed that these recombinant proteins could specifically bind to polyclonal antibody.4.The protein levels of SAGfm1 and SAGfm2 were verified by Western blot analysis and immunofluorescence localization.Western blot analysis result indicated that the protein levels of SAGs in the sensitive strains was significantly down regulated,but that in the resistant strains were up regulated.The results of immunofluorescence localization showed that SAGs were evenly distributed on the surface of the second-generation merozoites.Compared with the control group,the fluorescence signal intensity of SAGs in the sensitive strain treated with Ethanamizuril was significantly weakened,while that in the resistant strain was significantly enhanced.In conclusion,we speculate that these two proteins might not only be the key molecules of drug action,but also be related to drug resistance.This study is helpful to further understand the mechanism of anticoccidial drugs and delay the emergence of drug resistance.