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

Biao Zhou,Yigang Tong,Huan Zhou,Songying Ouyang,Chenpeng Fan,Feiyang Zhao,Vanja Perčulija,Zhiqiang Mi,Xiangkai Zhen,Gongyi Zhang

doi:10.1371/journal.ppat.1008394

Abstract

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.

Highlights

Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that commonly inhabits oral cavity, lower intestinal tract, and vaginal tract of healthy humans or other mammals [1]
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

Summary

Introduction

Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that commonly inhabits oral cavity, lower intestinal tract, and vaginal tract of healthy humans or other mammals [1]. Endolysins are hydrolytic enzymes that recognize the host cell-wall and cleave the peptidoglycan to digest the bacterial cell-wall for release of phage progeny during the lytic cycle [7, 8], which is why they hold promise for the treatment of multidrug-resistant bacterial infections as an alternative to antibiotics [9,10,11]. The modular structure of lysins makes it possible to design bioengineered endolysins that have desired properties, such as higher activity, or broader killing spectrum. Chimeric endolysins such as ClyH and ClyJ are engineered to fight against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae (MRSP) [23, 24]

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