Eimeria tenella: specific EtAMA1-binding peptides inhibit sporozoite entry into host cells
Eimeria tenella: specific EtAMA1-binding peptides inhibit sporozoite entry into host cells
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DOI：
10.3382/ps/pez298
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发表时间：
2019-10-01
期刊：
影响因子：
4.4
通讯作者：
Dalloul, Rami A.
中科院分区：
文献类型：
--
作者：
Ma, Dexing;Huang, Yuchen;Dalloul, Rami A.
由艾美耳球虫引起的禽球虫病给家禽业造成了巨大的经济损失。药物和活疫苗的应用在病原体耐药性和成本方面提出了特殊的挑战，因此需要替代的抗球虫策略。在这项研究中，从噬菌体展示肽库中筛选特异性结合E. tenella AMA1 （EtAMA1）的肽。4轮生物筛选后，采用酶联免疫吸附法（ELISA）对阳性噬菌体克隆进行鉴定。采用ELISA法检测两种多肽与EtAMA1和孢子子蛋白的结合能力。体外研究了两种靶肽抑制孢子虫侵袭MDBK细胞的作用，动物实验研究了两种噬菌体的抗球虫作用。基于TgAMA1晶体模板（PDB ID: 2 × 2Z）构建了EtAMA1胞外结构域（EctoAMA1）蛋白的三维（3D）结构模型，并分析了靶肽与EctoAMA1模型之间的分子对接。结果表明，两种噬菌体与EctoAMA1和孢子子蛋白有较强的相互作用。两个相应的特异性etama1结合肽（L和C）对抑制MDBK细胞的孢子子侵袭有显著作用。口服两种噬菌体的鸡对同源侵染有部分保护作用。同源性建模分析显示，EctoAMA1模型顶部形成了一个顶端疏水槽。分子对接表明EctoAMA1蛋白与这两种多肽之间存在相互作用，主要表现为疏水相互作用和分子间氢键的形成。上述结果表明，肽L和C，特别是L肽与E. tenella rhotry neck protein 2 （EtRON2）竞争结合到位于孢子体表面的EtAMA1上，从而抑制了寄生虫对细胞的侵袭。
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 x 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.