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

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Bacteriophage-derived endolysins are explored as alternatives to antibiotics for combating drug-resistant bacteria.
• The narrow spectrum of activity limits the clinical application of many endolysins.
• LysIME-EF1, an endolysin from Enterococcus faecalis phages, demonstrates potent bactericidal effects against E. faecalis strains.

Purpose of the Study:

• To investigate the structural basis of LysIME-EF1's lytic activity.
• To elucidate the role of a specific 8 kDa fragment in LysIME-EF1 function.
• To understand the molecular mechanism underlying LysIME-EF1's efficacy against E. faecalis.

Main Methods:

• X-ray crystallography was used to determine the 1.75 Å structure of LysIME-EF1.
• Site-directed mutagenesis and lytic activity assays were performed.
• In vivo animal infection models were utilized to assess efficacy.

Main Results:

• LysIME-EF1 forms a unique tetrameric structure with additional C-terminal cell-wall binding domains (CBDs).
• An internal ribosomal binding site and alternative start codon explain the translation of the 8 kDa CBD fragment.
• Mutagenesis and functional assays confirmed the importance of these additional CBDs for LysIME-EF1's architecture and lytic activity.

Conclusions:

• This study presents the first determined structure of a multimeric endolysin encoded by a single gene in E. faecalis phages.
• The unique multimeric structure and the role of additional CBDs are crucial for LysIME-EF1's potent lytic activity.
• Findings offer valuable insights for designing novel endolysins against the opportunistic pathogen E. faecalis.