Abstract
Avian coccidiosis caused by Eimeria leads to huge economic losses on the global poultry industry. In this study, microneme adhesive repeat regions (MARR) bc1 of E. tenella microneme protein 3 (EtMIC3-bc1) was used as ligand, and peptides binding to EtMIC3 were screened from a phage display peptide library. The positive phage clones were checked by enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was applied to further verify the binding capability between the positive phages and recombinant EtMIC3-bc1 protein or sporozoites protein. The inhibitory effects of target peptides on sporozoites invasion of MDBK cells were measured in vitro. Chickens were orally administrated with target positive phages and the protective effects against homologous challenge were evaluated. The model of three-dimensional (3D) structure for EtMIC3-bc1 was conducted, and molecular docking between target peptides and EtMIC3-bc1 model was analyzed. The results demonstrated that three selected positive phages specifically bind to EtMIC3-bc1 protein. The three peptides A, D and W effectively inhibited invasion of MDBK cells by sporozoites, showing inhibited ratio of 71.8%, 54.6% and 20.8%, respectively. Chickens in the group orally inoculated with phages A displayed more protective efficacies against homologous challenge than other groups. Molecular docking showed that amino acids in three peptides, especially in peptide A, insert into the hydrophobic groove of EtMIC3-bc1 protein, and bind to EtMIC3-bc1 through intermolecular hydrogen bonds. Taken together, the results suggest EtMIC3-binding peptides inhibit sporozoites entry into host cells. This study provides new idea for exploring novel strategies against coccidiosis.
Highlights
The phylum apicomplexa includes several well-known unicellular protozoan parasites such as Plasmodium, Toxoplasma, Neospora and Eimeria, all of which infect human or animals
Screening of phages binding to E. tenella microneme protein 3 (EtMIC3)‐bc1 The plaque forming unit per milliliter of the positive phages binding to EtMIC3-bc1 protein were identified after four rounds of affinity screening (Table 1), Table 1 Titer of phages after four rounds of biopanning and amplification
Invasion for apicomplexan protozoan parasites was initiated based on the interplay between the apex of parasites and host cells, during which a series of secreted proteins at the parasite-host interface are indispensable for the parasites to penetrate into host cells
Summary
The phylum apicomplexa includes several well-known unicellular protozoan parasites such as Plasmodium, Toxoplasma, Neospora and Eimeria, all of which infect human or animals. Previous studies demonstrated that EtMIC3 effectively facilitates the invasion of sporozoites into host cells by recognizing sialylated glycans, and is responsible for guiding E. tenella sporozoites to the invasion sites in chicken gut [12, 13]. Taking into account that EtMIC3 protein plays key roles in the process of sporozoites invasion of host cells, we postulated that peptides binding to EtMIC3 protein could effectively inhibit invasion of cells by E. tenella sporozoites. With the aim to verify the above hypothesis and explore novel strategies against coccidiosis, in the present study, the recombinant MARb and one of the four repeated MARc domains of EtMIC3 protein (EtMIC3-bc1) were selected as ligand, and peptides binding to EtMIC3 were screened from phage display peptide library. The model of three-dimensional (3D) structure for EtMIC3-bc protein was conducted, and molecular docking between target peptides and EtMIC3-bc model was analyzed
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