Comparative Proteomics And Immuno-Proteomics Of Eimeria Tenella

Eimeria tenella is probably the most important species of Eimeria that infects chickens and causes a decrease in weight gain and feed consumption accompanied by bloody diarrhea, resulting generally in the death of the chickens and leading to high economic losses for the poultry industry.Some of specific proteins of E. tenella play an important role in the parasite life cycle. Post-genomic studies will make it possible to understand the complexity of the parasites and their interactions with host cells. Here we present a comparable systematic reference map of the proteins from E. tenella unsporulated and sporulated oocysts. The proteins expressed at the two stages were resolved between isoelectric points between pH 3-10 and displayed with two-dimensional polyacrylamide gel electrophoresis. Gels were stained with Coomassie Blue G250 and analysied using ImageMasterTM. Protein spots were found to have changes in abundance and quantity during development. we anlysed 18 commom spots in two stages; 15 spots only existed in unsporulated stage and 21 spots only in sporulated stage and identified these spots using MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and MALDI-TOF-TOF-MS. The data were searched against NCBI database and E. tenella protein sequence database respectively.43 proteins were identified as protein of E. tenella and homologous proteins to other Apicomplexa parasites or protozoan. Among of them, the abundance of Enolase, actin depolymerizing factor, large subunit ribosomal protein L23 and 19KDa sporozoite antigen in unsporulated oocysts stage showed lower than in sporulated oocysts stage, the abundance of Lactate dehydrogenase showed higher than sporulated oocysts stage inversely, the abundance of the rest proteins didn't show visiblely change. Proteins showed only in unsporulated oocysts stage including Pyruvate kinase, hypothetical protein, Mannitol-1-phosphatase, aspartyl proteinase and 14-3-3 protein of E. tenella and 7 homologous proteins of other other Apicomplexa parasites or protozoan, included putative secretory protein of Theileria annulata, eukaryotic translation initiation factor 5 of Toxoplasma gondii, polyadenylate-binding protein 2 of Tetrahymena thermophila SB210, heat shock protein (E. acervulina), hypothetical protein PVX₀91055 of Plasmodium vivax, 6-phosphofructokinase, putative of Plasmodium vivax, proteasome subunit of Cryptosporidium hominis TU502 and so on.. Same to unsporulated oocysts stage, the proteins of sporulated oocysts were identified including sporozoite antigen and 4 homologous proteins of other Apicomplexa parasites or protozoan, they are included hypothetical protein, cytoplasmic dynein heavy chain 2 protein and MSP domain containing protein of Tetrahymena thermophila SB210.Two-dimensional electrophoresis (2-DE) was employed to analyse the solubilised proteins of sporulated oocysts and the second-generation merozoite of Eimeria tenella; Here we present a systematic reference map of the proteins from above two stages. Gels were stained and analyzed by image analysis software. About 678 and 660 spots were detected in the gels of the two stages,14 spots keep stable volume%. After analyzed and searched,8 spots were identified including lactate dehydrogenase,19KD sporozoite antigen, Mic2, enolase and actin depolymerizing factor of E.tenella and an homologous protein of proteasome subunit of Theileria annulata.In order to understand the mechanisms of immune responses, we describe our work using immuneoproteomic approach, a method combining the specificity of antibody which produced by host, to screen new antigenic proteins from the different stages in life cycle of E.tenella.Immunoproteomic assay was used to identify antigenic proteins from the total proteins of unsporulated oocysts of Eimeria tenella.101 protein spots were recognized by chicken sera infected naturally with E. tenella. After 46 spots were analyzed and searched,14 and 17 spots were identified as proteins of E.tenella and homologous proteins to other apicomplexa parasites or Paramecium tetraurelia and Tetrahymena thermophila These proteins included lactate dehydrogenase, enolase, sporozoite antigen, actin depolymerizing factor, immunoglobulin heavy chain binding protein, pyruvate kinase (PK), tubulin beta chain (Beta-tubulin), hypothetical protein of E. tenella and heat shock protein of Eimeria acervulina, several hypothetical proteins of Paramecium tetraurelia, 6-phosphofructokinase and an hypothetical protein of Plasmodium vivax, PfmpC and an hypothetical protein of Plasmodium falciparum 3D7, polyadenylate-binding protein 2 and several hypothetical proteins of Tetrahymena thermophila SB210. The rest proteins were searched against the E. tenella protein sequencedatabase using'Mascot in-house'(version 2.1) search engine in automated mode and 12 unknown presumed proteins were identified. After the amino acid sequence of the unknown proteins were searched using BLAST against non-redundant sequence databases (NCBI),9 homologous proteins were found homologous to proteins of other apicomplexa parasites. They were glyceraldehyde-3-phosphate dehydrogenase, eukaryotic translation initiation factor 5 and myosin-A of Toxoplasma gondii, SNF2 family N-terminal domain containing protein of Plasmodium vivax, myosin B and fumarate hydratase of Babesia bovis, putative secretory protein of Theileria annulata, pyruvate dehydrogenase El component beta subunit, mitochondrial of Theileria parva and proteasome subunit of Cryptosporidium hominis.Immunoproteomic assay was used to identify antigenic proteins from the total proteins of sporulated oocysts with anti-E.tenella chicken serum of Eimeria tenella. Approximately 80 protein spots were respectively recognized by chicken sera infected artificially with E. tenella. Fourty spots were analyzed by MALDI-TOF-MS and MALDI-TOF-TOF-MS and searched through NCBI database using'Mascot'server.22 and 8 spots were identified as proteins of E. tenella and homologous proteins to other apicomplexa parasites or Paramecium tetraurelia and Tetrahymena thermophila. These proteins included:sporozoite antigen, enolase, lactate dehydrogenase, tubulin beta chain, transhydrogenase, Microneme protein MIC3, unknown protein,14-3-3 protein, surface antigen 10, enoyl-acyl carrier reductase of E. tenella and unknown protein RB1-a of Eimeria acervulina, dihydrofolate reductase-thymidylate synthase and conserved hypothetical protein of Cryptosporidium parvum Iowa II, several hypothetical proteins of Paramecium tetraurelia strain d4-2 and Tetrahymena thermophila SB210. The rest proteins of this stage were searched against the E. tenella protein sequence database using'Mascot in-house'(version 2.1) search engine in automated mode,8 unknown proteins were identified. After the amino acid sequence of the unknown proteins were searched using BLAST against non-redundant sequence databases (NCBI),3 homologous proteins were identified as AAA family ATPase of Plasmodium vivax, myosin-A of Toxoplasma gondii and an hypothetical protein of Trichoplax adhaerens.Whole resolved proteins of the second-generation merozoite of E. tenella were analyzed by two-dimensional gel electrophoresis (2-DE) and western blot using the chicken sera infected artificially with E. tenella. Approximately 640 spots were detected on proteome map of the second generation merozoite stained by Coomassie brilliant blue G-250 and 85 spots were recognized on western blot map as antigens. Forty four spots of the antigens were identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) and MALDI-TOF-TOF-MS. Twenty six proteins of E. tenella and 3 homologous proteins to other apicomplexan parasites and protozoan were identified using 'Mascot'server. These proteins included lactate dehydrogenase, enolase,14-3-3 protein, microneme proteins, tubulin beta chain, SERPIN1 protein precursor, large subunit ribosomal protein L23, surface antigens of E. tenella, heat shock protein (HSP70) of Eimeria acervulina protein phosphatase type 1 of Toxoplasma gondii and hypothetical protein GSPATT00020155001 of Paramecium tetraurelia. The rest proteins were searched against the E. tenella protein sequence database using'Mascot in-house'(version 2.1) search engine in automated mode and 11 unknown proteins were identified. After the amino acid sequence of the unknown proteins were searched using BLAST against non-redundant sequence databases (NCBI), a surface antigen 12 of E. tenella and 6 proteins homologous to other apicomplexa parasites were found. They were membrane skeleton protein IMC2A, mitochondrial F1-ATP synthase beta subunit precursor, 3-oxoacyl-acyl-carrier-protein synthase and catalase of Toxoplasma gondii, Vps26 of Plasmodium falciparum 3D7, and hypothetical protein TRIADDRAFT₆0424 of Trichoplax adhaerens.