肺炎克雷伯菌噬菌体KP-C01识别多种宿主表面受体的分子机制

噬菌体结构和遗传多样性及宿主受体的复杂性决定了二者的互作机制必然是多样且复杂的。本项目前期研究中发现，肺炎克雷伯菌噬菌体KP-C01识别细菌8110W-B的多糖和噬菌体耐受菌C01R-122R的蛋白受体，而与其相似性极高的KP100仅识别前者，但具体受体和分子机制仍不清楚。初步判定KP-C01和KP100中ORF48编码的尾针蛋白为其各自的受体结合蛋白（RBP），推测二者之间的某些差异导致了其识别受体的不同。本研究将通过原核表达和竞争吸附确定两株噬菌体的RBP；对亲本株和耐受噬菌体突变株进行差异基因组、蛋白质组分析及pull down实验确定两株噬菌体识别的宿主受体，并利用基因敲除与回补进行验证；根据同源建模和活性预测结果，表达RBP的不同截短及单位点突变蛋白确定其识别受体的关键结构域和氨基酸位点。本研究对丰富噬菌体与宿主的互作机制具有重要意义，也为改造噬菌体以拓宽其裂解谱提供理论依据。

The diversity of phages structure and genetic and the complexity of host receptors determine that the interaction mechanisms between phages and host must be diverse and complex. In the previous study of this project, it was found that Klebsiella pneumoniae phage KP-C01 could recognize the polysaccharide receptor of bacteria 8110W-B and the protein receptor of phage-resistant bacteria C01R-122R, while KP100, which is very similar to it, only recognized the former receptor, but the specific receptors and mechanism of action was still unclear. It was preliminarily determined that the tail spike encoded by ORF48 in KP-C01 and KP100 were their respective receptor binding proteins (RBPs) and it was speculated that some differences between the two RBPs led them to recognize different host receptors. In this study, RBP in two phages was determined by prokaryotic expression and competitive adsorption experiment. Differentially genomic, proteomic and pull down analysis were performed on the wild-type strains and the phage-resistant mutant strains to determine the host receptors recognized by the two phages, and gene knockout and complement were used for verification. Based on homology modeling and activity prediction, different truncated proteins and unit point mutations of RBP were expressed to identify the key domains and amino acid sites of the receptor recognition. This study is of great significance for enriching the interaction mechanism between phages and host, and also provides a theoretical basis for modifying phage to broaden its lytic spectrum.