Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage
Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage
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粪肠球菌噬菌体中单个基因编码的新型双组分内溶素的结构和功能见解
DOI：
10.1371/journal.ppat.1008394
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发表时间：
2020
期刊：
影响因子：
6.7
通讯作者：
Songying Ouyang
中科院分区：
文献类型：
--
作者：
Biao Zhou;Xiangkai Zhen;Huan Zhou;Feiyang Zhao;Chenpen Fan;Vanja Perčulija;Yigang Tong;Zhiqiang Mi;Songying Ouyang
使用噬菌体衍生的内溶素作为对抗耐药细菌的替代策略最近重新引起了人们的兴趣。然而，它们的应用仍然受到其狭窄的活性谱的阻碍。在我们以前的工作中，我们证明了内溶素LysIME-EF 1对多种粪肠球菌（Enterococcus faecalis，E.粪便）。在此，我们观察到一个8 kDa的片段，并假设它有助于LysIME-EF 1裂解活性。为了检验我们的假设，我们在1.75 μ m分辨率下确定了LysIME-EF 1的结构。LysIME-EF 1具有独特的结构，其中一个全长LysIME-EF 1与三个额外的C-末端细胞壁结合结构域（CBD）形成四聚体，所述三个额外的C-末端细胞壁结合结构域对应于上述8 kDa片段。此外，我们确定了一个内部的核糖体结合位点（RBS）和LysIME-EF 1基因内的替代起始密码子，这被证明是负责截短的CBD的翻译。为了阐明LysIME-EF 1裂解活性的分子机制，我们结合诱变，裂解活性测定和体内动物感染实验。结果证实，额外的LysIME-EF 1 CBD对于LysIME-EF 1结构及其裂解活性是重要的。据我们所知，这是第一个确定的结构，多聚体内溶素编码的一个单一的基因在大肠杆菌。粪菌因此，它可以为设计针对机会致病菌E的有效内溶素提供有价值的见解。粪便。
Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.