Eimeria tenella: specific EtAMA1-binding peptides inhibit sporozoite entry into host cells.

Avian coccidiosis caused by Eimeria inflicts high economic losses to the poultry industry. Application of drugs and live vaccines presents particular challenges in pathogen resistance and cost, hence alternative anti-coccidial strategies are needed. In this study, peptides that specifically bind E. tenella AMA1 (EtAMA1) were screened from a phage display peptide library. The positive clones of target phages were characterized by ELISA after four rounds of biopanning. The binding capabilities with EtAMA1 and sporozoite proteins for the two selected peptides were detected by ELISA. The role of the two target peptides in inhibiting sporozoite invasion of MDBK cells was evaluated in vitro and the anti-coccidial effects of the two phages were assessed by an animal experiment. The three-dimensional (3D) structural model of EtAMA1 extracellular domain (EctoAMA1) protein was constructed based on the crystal template of TgAMA1 (PDB ID: 2 × 2Z), and the molecular docking between target peptides and EctoAMA1 model was analyzed. The results showed that two selected phages strongly interacted with EctoAMA1 and sporozoites protein. Two corresponding specific EtAMA1-binding peptide (named L and C) showed significant effects on inhibiting sporozoite invasion of MDBK cells. Chickens orally fed the two target phages showed partial protection against homologous challenge. Homology modeling analysis showed an apical hydrophobic groove was shaped on the top of the EctoAMA1 model. Molecular docking indicated the interaction between the EctoAMA1 protein and the two peptides, which was mainly reflected by the hydrophobic interaction and formation of intermolecular hydrogen bond. The above results suggest that the peptides L and C, especially L peptide, competed with E. tenella rhotry neck protein 2 (EtRON2) for binding to EtAMA1 located on the surface of sporozoites, and therefore inhibited the parasite invasion into cells.