Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage

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 angstrom 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. Author summary LysIME-EF1, an endolysin that lyses E. faecalis, displays the prospect of treating E. faecalis infection. We find that the C-terminal cell-wall binding domain (CBD) is important for the lytic activity of LysIME-EF1. By determining the crystal structures of wild type (WT) LysIME-EF1 and its C-terminal CBD, this study reveals how the holoenzyme is organized to carry out its highly efficient lytic activity. Our finding provides structural and functional evidence that LysIME-EF1 belongs to a unique two-component multimeric endolysin encoded by a single gene.