The Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China.
Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China.
PLoS Pathog. 2020 Mar 16;16(3):e1008394. doi: 10.1371/journal.ppat.1008394. eCollection 2020 Mar.
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.
利用噬菌体衍生的内溶素作为对抗耐药菌的一种替代策略，最近重新引起了人们的兴趣。然而，它们的应用仍然受到其活性谱狭窄的限制。在我们之前的工作中，我们证明了内溶素 LysIME-EF1 对多种粪肠球菌（E. faecalis）菌株具有有效的杀菌活性。在这里，我们观察到一个 8 kDa 的片段，并假设它有助于 LysIME-EF1 的溶菌活性。为了检验我们的假设，我们确定了 LysIME-EF1 在 1.75 Å 分辨率下的结构。LysIME-EF1 呈现出独特的结构，其中一个全长的 LysIME-EF1 形成一个四聚体，另外还有三个额外的 C 端细胞壁结合结构域（CBD），对应于上述 8 kDa 的片段。此外，我们在 LysIME-EF1 基因中鉴定出一个内部核糖体结合位点（RBS）和一个替代起始密码子，证明它们负责翻译截短的 CBD。为了阐明 LysIME-EF1 的溶菌活性的分子机制，我们结合了突变、溶菌活性测定和体内动物感染实验。结果证实，额外的 LysIME-EF1 CBD 对于 LysIME-EF1 的结构和其溶菌活性是重要的。据我们所知，这是第一个在粪肠球菌噬菌体中由单个基因编码的多聚体内溶素的确定结构。因此，它可能为设计针对机会性病原体粪肠球菌的有效内溶素提供有价值的见解。
