肠炎沙门氏菌噬菌体尾丝受体结合蛋白的结构解析及其在宿主感染识别中的分子机制

Salmonella is a kind of important food borne pathogens. With the emergence of drug-resistant bacteria and the frequent occurrence of food poisoning, bacteriophage has become an antibacterial candidate. Due to the large number of Salmonella serotypes and the specificity of the phage, how to broaden the phage host spectrum has become the focus of research. The studies on model phages showed that the receptor binding protein (RBP) played an important role in the process of phage-host infection and recognition. We have obtained that the Salmonella phage which belongs to the FelixO1 phage family, so far there is no report on the study of the RBP of this family. The study plans to express the related tail fiber proteins, and fix the position of RBP cluster gene in whole genome; then, according to the well identification RBP cluster gene and its amino acid sequence, we will construct and identify different recombinant phages which will contain different mutant regions of the RBP gene by homologous recombination, then analyze the function of the RBP mutation sites in phage recognition. Further, we will comparative analysis RBP gene expression in the transcription level and its impact on the expression of receptor protein in protein level, respectively. Finally, we clarify the molecular mechanism of RBP in phage recognition. This study will provide a way to broaden the phage host spectrum, and also provided technical supports for phage control safety and effectively.

沙门氏菌是一种重要的食源性病原菌，随着耐药菌不断出现以及食物中毒事件频发，噬菌体已成为抗菌候选。由于沙门氏菌血清型众多，而噬菌体的种属特异性，如何拓宽噬菌体宿主谱已成为研究焦点。对模式噬菌体的研究表明，噬菌体尾丝受体结合蛋白(RBP)在宿主感染识别中发挥首要作用。我们获得的沙门氏菌噬菌体PA13076属于FelixO1家族，目前尚未有该家族噬菌体RBP的研究报道，其在噬菌体感染识别中的分子机制亦不清楚。本研究拟对噬菌体PA13076的RBP进行基因簇定位及功能鉴定，明确编码RBP的关键基因簇并鉴定其与宿主的相互作用特性；通过同源重组构建并鉴定携带不同RBP片段的重组噬菌体，分析重组噬菌体在宿主感染识别中的不同RBP基因和蛋白的表达；转录分析感染宿主受体蛋白OmpC表达情况，明确RBP在噬菌体-宿主感染识别中的分子机制，为噬菌体扩谱改造奠定基础，亦为噬菌体防控沙门氏菌污染提供技术支撑。

噬菌体尾丝受体结合蛋白(RBP)在宿主感染识别中发挥首要作用。针对本研究室分离的沙门氏菌噬菌体PA13076，经全基因组测序分析发现，该噬菌体基因组序列较为独特，目前尚未有该家族噬菌体RBP的研究报道，其在噬菌体感染识别中的分子机制亦不清楚。本研究以沙门氏菌噬菌体PA13076为研究对象，首先，对该噬菌体进行基因组测序和基因功能注释，ORF8，ORF9，ORF64为尾部蛋白，ORF62、ORF63为尾部纤维组装蛋白，其次，通过次级感染法和双层平板法筛选出1株沙门氏菌噬菌体抗性菌（R3），通过将噬菌体抗性菌R3、原宿主菌ATCC13076与原沙门氏菌噬菌体PA13076共培养，并反复传代20次以上，筛选出1株突变噬菌体BPR3，该突变噬菌体较原噬菌体拓宽了宿主谱，可以同时裂解原宿主菌ATCC13076和噬菌体抗性菌R3，从而为后期噬菌体尾丝受体结合蛋白的关键基因簇的研究提供了依据；然后，将突变噬菌体进行全基因组测序，与原噬菌体相比，突变噬菌体的尾丝相关蛋白（ORF8）在基因序列和氨基酸序列上有8个位点突变，尾丝蛋白（ORF9）上有两个位点突变，其他尾丝相关蛋白编码基因未突变；随后，克隆表达含有GST标签的135 KDa (1G8和RG8)和47 KDa (G9和RG9)重组融合蛋白；通过吸附实验，重组蛋白1G8和RG8对噬菌体PA13076吸附率影响不大，但是1G9和RG9对噬菌体的吸附率影响显著，噬菌体尾丝受体结合蛋白G9为该噬菌体的主要尾丝受体结合蛋白RBP，尾部蛋白G8可能为基盘蛋白。与原噬菌体PA13076相比，突变噬菌体尾丝受体结合蛋白G9共有2个单核苷酸点突变，导致其编码的氨基酸分别从Gly（G）变为Glu（E），Asp（D）变为Gly（G），研究明确了该噬菌体参与宿主识别的RBP的编码区及氨基酸序列，解析RBP在噬菌体-宿主识别中的分子机制。