Study On The Biological Function Of Apical Membrane Antigens In Eimeria Tenella Host Cell Invasion

Coccidiosis is an intestinal disease caused by infection in the Eimeria spp.,which causes severe economic losses to the poultry industry worldwide.At present,the main methods for coccidiosis are anticoccidia drugs and vaccines.However,due to the development of anticoccidial drug resistance and the high cost of vaccine production,Therefore,new control strategies need to be developed.Invasion of sporozoites and merozoites into host cells is the key to the establishment of Eimeria infection,and blocking the invasion of Eimeria is an effective method to control coccidiosis.The apical membrane antigen is a highly conserved micronme protein in apicomplexan parasites,which plays an important role in the process of invading host cells.Recent studies have found that E.tenella has four AMA paralogues?AMA1-4?.In previous research,we found that E.tenella AMA1?EtAMA1??ToxoDB Accesion No.:ETH₀0007745?is a key protein for sporozoite invasion of the host and can be interact with the conserved Hypothetical proein 317?EtCHP317?and rhoptry neck protein 2?EtRON2?,but the specific mechanism is not clear.This study intends to further study the characteristics and biological functions of EtAMA1.At the same time,the characteristics and biological functions of two paralogues of EtAMA1,EtAMA2?ETH₀00048600?and EtAMA3?ETH₀0017730?were preliminary studied.The research results laid the foundation for elucidating the function and mechanism of AMAs proteins during Eimeria host cell invasion.1.Further investigation of characteristics and biological function of E.tenella apical membrane antigen 1In order to further study the characteristics and functions of E.tenella apical membrane antigen 1,further analysis was performed using methods such as secretion test,indirect immunofluorescence colocalization,invasion test,and iTRAQ analysis.The results show that the protein is secreted by the microneme and is regulated by temperature and fetal calf serum?FCS?.The distribution of EtAMA1,EtCHP317 and EtRON2 is basically the same.The inhibition rates of sporozoite invasion in EtAMA1,EtCHP317 and EtRON2 were 40.9%,33.1% and 41.1%,respectively.However,the inhibition rate of sporozoites invasion after mixing anti-EtAMA1 and anti-EtCHP317,anti-EtAMA1 and anti-EtRON2 at a ratio of 1: 1 was only 21% and 21.3%.After transfection of the recombinant plasmid pcDNA3.1-?+?-flag-Et AMA1 into DF-1 cells,the invasion rate was significantly higher than that of the blank control group.iTRAQ identified 163 differential proteins,including 91 up-regulated proteins and 72 down-regulated proteins.The results provide a basis for elucidation of the molecular mechanism by which EtAMA1 plays an important role in E.tenella invasion of host cells.2.Characteristics and biological function of apical membrane antigen 2 of E.tenellaIn order to study the molecular characteristics and biological function of apical membrane antigen 2 of E.tenella?EtAMA2?,indirect immunofluorescence assay,invasion inhibition assay,quantitative real-time PCR,western blotting and secretion assays were used to study its biological properties.The results indicated that EtAMA2 protein is a type I integral membrane protein with a transmembrane region and a signal peptide.Compared with other EtAMAs,EtAMA2 has the same number?10?of cysteine residues in the structural domains DI and DII,and the same number?6?of cysteine residues in DIII with that of EtAMA1,with five in the same position and one different.The transcriptional level of EtAMA2 was higher in sporozoites and second-generation merozoites than in unsporulated oocysts and sporulated oocysts,but the protein was expressed only in second-generation merozoites.EtAMA2 was mainly distributed on the cytoplasm of free sporozoites and second-generation merozoites,at the apical end of intracellular sporozoites.EtAMA2 is secreted by the microneme and its secretion is regulated by temperature.The anti-rEtAMA2 polyclonal antibody had no significant effect on sporozoites invasion into host cells.After transient transfection of EtAMA2 into DF-1 cells,there was no significant difference in sporozoite invasion rate compared with the blank control group.Screening of second-generation merozoites invading host cells revealed that they can invade CEF and MDCK cells.The results show that Et AMA2 is a merozoite stage regulatory protein,which has no significant effect on the invasion of sporozoites into host cells,and its function in merozoites needs further study.3.Characteristics and biological function of apical membrane antigen 3 of E.tenellaIn order to study the molecular characteristics and biological function of apical membrane antigen 3 of E.tenella?EtAMA3?,indirect immunofluorescence assay,invasion inhibition assay,quantitative real-time PCR,western blotting and secretion assays were used to study its biological properties.The results indicated that EtAMA3 protein is a type I integral membrane protein and has a transmembrane region,a signal peptide and has 24.31%-38.45% homology with other EtAMAs.Compared with other EtAMAs,EtAMA3 had the same number?10?of cysteine residues in the domains DI and DII,but six cysteine residues found in EtAMA1 DIII were lacking in EtAMA3 DIII.Its transcription level was the highest in sporozoites,the lowest transcription levels were found in the unsporulated oocysts and second-generation merozoites,but the protein was expressed only in sporozoites and sporulated oocysts.EtAMA3 was mainly distributed on the cytoplasm of free sporozoites,at the apical end of intracellular sporozoites.EtAMA3 is secreted by the microneme and its secretion is regulated by FCS.The anti-rEtAMA3 polyclonal antibody had significant effect on sporozoite invasion into host cells at the concentration of 300 ?g/ mL.After transient transfection of EtAMA3 into DF-1 cells,there was no significant difference in sporozoite invasion rate compared with the blank control group.The results showed that EtAMA3 is a sporozoite stage regulatory protein and plays an important role in sporozoite invasion of host cells.