发酵乳杆菌温和噬菌体ΦYB5裂解素和穿孔素的结构功能解析

Bacteriophage infections of lactic acid bacteria are a serious risk in the dairy industry, which can lead to slow lactic acid production or even the total failure of fermentation. However, the bacteriophage infections resulted in the release of the intracellular enzymes such as peptidases and lipases from the infected strains improving the texture and flavour development in cheese ripening. Considering the significance of bacteriophages in the dairy fermentation, it is necessary to deeply investigate the lysis mechanisms of the bacteriophages. In this project, the endolysin and holin (Lyb5-Hyb5) cassette, the two main functional proteins for the lysis were identified from the temperate bacteriophage ΦYB5 in Lactobacillus fermentum YB5 and their structures and functions will be analysized through bioinformatic tools, biochemistrial and molecular techniques. Firstly, the N-terminus of Lyb5 will be deleted and expressed in E.coli and Lactococcus lactis. As a result, the transmenbrane style of Lyb5 will be found out by determination of the migration length of the Lyb5 and its mutants in Zymogram-PAGE gel and of the enzyme activities in cytoplasm, periplasm and menbrance. Secondly, to isolate the antiholin gene on the genome of bacteriophage which is dedicated to triggering the cell lysis, the potential open reading frames (ORF) located in the hyb5 gene or upstream/downstream will be coexpressed with Hyb5, the delay time of the lysis cells will be determined, and the mechanism for exquisitely programmed the lysis trigger time of antiholin will be developed. The hole formation model of Hyb5 will be characterized by the observation of the chimeras and oligomerization of the holin Hyb5 in the membrane by chemical cross-linking,Western blot and electron microscope. Consequently, the cooperation of Lyb5 and Hyb5, as well as the formation of phaques and one-step growth curves of mutant ΦYB5Δhyb5 or ΦYB5Δantiholin will be detected so that a genetic programmed timing model for cell lysis caused by ΦPYB5 will be set up. As summary, the aim of this project is to explore the roles of Lyb5 and Hyb5 in triggering the cell lysis and provide a rational theory to achieve the control of phage infection and to construct the lysis system for lactic acid bacteria starters.

乳制品发酵中噬菌体感染生产菌株，导致产酸力降低甚至发酵失败，但细胞破裂的同时伴随着胞内酶的释放，有利于发酵乳后期成熟及风味物质形成。基于此，如何控制和有效利用噬菌体，其本质是对裂解机制的深入认识。课题组分离到一株发酵乳杆菌温和噬菌体ΦYB5，其基因组上编码有裂解素和穿孔素基因，本项目拟在前期工作的基础上，借助生物信息学、分子生物学和生物化学等技术，对此裂解素和穿孔素结构进行功能解析。通过缺失突变和定点突变，确定裂解素的分泌方式；通过检测穿孔素与其基因内外潜在阅读框的共表达对裂菌时间的延迟，定位调节基因并阐明其裂解触发调节机制；利用体外化学交联、Western blot、原子力显微镜观测等手段，分析穿孔素在细胞内、外的聚集特性，提出膜通道形成模式；探究裂解素和穿孔素破裂细胞的协同作用并揭示其裂解机制，从而为有效防止噬菌体感染及对噬菌体裂解系统的改造、建立高效的乳酸菌裂解表达系统提供理论基础。

乳制品发酵过程中噬菌体感染生产菌株，造成产酸能力能力下降进而影响产品质量。本课题首先调查了国内酸奶生产中噬菌体的生态多样性，结果发现很多生产菌株以溶原状态存在，这些菌株经丝裂霉素C、紫外线诱导后，获得了6株乳杆菌温和噬菌体，其中包括1株德氏乳杆菌和1株发酵乳杆菌的噬菌体；比较基因组限制性内切酶图谱和外壳蛋白发现，这6株噬菌体基因组与已报道的乳杆菌噬菌体完全不同，说明国内乳品生产中噬菌体存在广泛且多样，对乳品发酵造成很大的威胁。同时还分离到1株德氏乳杆菌的烈性噬菌体phiLJB；phiJB与已报道的德氏乳杆菌噬菌体相似性较低，GC%高达47.7%，是一个快速进化的噬菌体。烈性噬菌体phiLJB编码的裂解素对常见腐败菌如Staphylococcus aureus 、Listeria monocytogenes有很强的抑制作用，可作为天然食品防腐剂。.细菌噬菌体基因组多为双链DNA，以二组分（裂解素-穿孔素）系统裂解细胞。以发酵乳杆菌温和噬菌体φPYB5基因组上的裂解盒（Lyb5-Hyb5）为例分析其分泌和裂解机制。裂解素Lyb5编码细胞壁水解酶，其 N-端携带有29aa的信号肽SPLyb5，通过构建Lyb5信号肽缺失突变体，比较Lyb5及突变体在大肠杆菌和乳酸乳球菌中表达后的细胞形态、生长速率及亚细胞定位，结果发现SPLyb5将Lyb5由胞质转运至周质并锚定在细胞膜上。以核酸酶Nuc为报告基因与SPLyb5形成嵌合体进一步证实了Lyb5分泌到周质空间，因此，说明Lyb5在SPLyb5引导下以非活性状态分泌并锚定在周至空间，其裂解释放受Hyb5调节。穿孔素Hyb5为膜蛋白，通过穿孔素Hyb5基因内部及上下游潜在的ORF与裂解盒共表达，发现Hyb5自身编码的ORF2具有裂解调控因子antiholin的作用。检测已知分子量大小递增报告基因与Hyb5共表达时细胞膜外的渗漏情况，确定Hyb5在细胞膜上形成的孔洞大小为针孔状；化学交联及凝胶层析分析，表明Hyb5在膜上形成四聚体。.根据上述结果，推测噬菌体φPYB5裂解细胞的方式：Lyb5在胞质中合成被运输至周质并以无活性锚定在细胞膜上，Hyb5在细胞膜上与ORF2相互作用；在特定时刻Hyb5与ORF2脱离，形成四聚体的小针孔，改变膜质子动力势进而激活并释放Lyb5，降解肽聚糖致使细胞破裂释放出新噬菌体颗粒。