gp38蛋白决定大肠杆菌噬菌体Bp7宽宿主谱的机制

The receptor-binding protein of bacteriophage and the receptors on the host cells determine the phage host range. Our preliminary studies demonstrated that bacteriophage Bp7 has broad host range, and gp38 was the receptor-binding protein of Bp7. However, the role of gp38 in determination of broad host range remains unclear. We hypothesized the receptor-binding domain of gp38 determined the broad host range of Bp7. To test hypothesis, expressed protein gp38 and E.coli K12 will be used in this study. The receptors on host cell recognized by gp38 will be confirmed by gene knock-out, pull-down assays, and mass spectrometry and so on. Then the receptor-deletion strains and trans-complementation strains will be constructed, and a series of truncated gp38 with different length will be expressed in prokaryotic system. The receptor-binding domains of gp38 to their relevant receptors will be determined by detecting the affinities with different receptor-deletion strains. The confirmed receptor-recognition domains will be synthesized in segment, and the binding ability of these segments to different receptors will be detected by western ligand affinity blot assay, to identify the key segments and their relevant receptor types. Then the relevance of receptors to receptor-binding domain and key segments of gp38 will be confirmed. Also, a number of E.coli clinical strains will be used to detect the consistence of Bp7 host range and strains recognized by receptor-binding domain peptides of gp38. This project will reveal the mechanism of gp38 caused bacteriophage Bp7 broad host range, and provide the theoretical basis for developing phage biological product.

噬菌体受体识别蛋白的特征及其识别受体的种类决定噬菌体的宿主谱。申请者前期研究发现gp38是宽宿主谱噬菌体Bp7的受体识别蛋白，但是gp38如何决定Bp7宽宿主谱的机制尚不明确。申请者以“gp38的受体结合域特征决定Bp7的宽宿主谱”为理论假说，以gp38蛋白和宿主菌E.coli K12为研究对象，利用基因敲除、pull-down、质谱分析等研究手段，筛选gp38识别的受体，构建宿主菌的各受体缺失株。表达不同长度的gp38截短体蛋白，与各受体缺失株作用，找出gp38的各受体结合域；分段合成各受体结合域的肽段，利用配基印迹技术，分析gp38识别各种受体的关键位点，最终明确gp38的受体结合域及关键位点与各受体的对应关系。在多株大肠杆菌临床株上检测gp38各受体结合域识别的菌株与Bp7宿主谱的一致性。通过以上研究，可阐明gp38决定噬菌体Bp7宽宿主谱的机制，为开发噬菌体生物制剂提供理论基础。

噬菌体受体识别蛋白的特征及其识别受体的种类决定噬菌体的宿主谱。项目以噬菌体尾丝蛋白gp38和宿主菌E.coli K12为研究对象，利用基因敲除、pull-down、质谱分析、噬斑检测等研究手段，筛选噬菌体Bp7识别的受体种类，分析尾丝蛋白gp38的关键受体结合位点，明确噬菌体Bp7与宿主菌E.coli K12的相互作用机制，并在临床株上进行验证。研究结果表明，噬菌体Bp7通过受体识别蛋白gp38识别宿主菌E.coli K12表面膜蛋白受体OmpC和LamB，与之可逆结合后，噬菌体进一步吸附到宿主菌E.coli K12表面的内核心多糖受体，并与之不可逆的结合，进而将核酸注入宿主菌内完成侵染过程。尾丝蛋白gp38识别受体的关键活性区域与其空间构象密切相关，主要集中在163-169aa, 187-189aa, 616-624aa以及C端244-263aa。噬菌体Bp7可以同时识别内核心多糖类受体和膜蛋白类受体，内核心多糖保守性较强，为大肠杆菌细胞壁的主要成分，OmpC和LamB作为膜蛋白也普遍存在于大肠杆菌表面，因此噬菌体Bp7能够有更高的几率吸附并裂解携带这些受体的大肠杆菌，从而表现出宽宿主谱的特征。通过本项目研究，阐明了 gp38宽宿主谱的分子机制，为开发宽宿主谱噬菌体制剂提供了理论基础。