Study On Three-dimensional Structure And Molecular Basis For Function Of Enterococcus Faecalis Phage Endolysin LysIME-EF1

Globally,as the abuse of antibiotics becomes more and more serious,more and more pathogens are exhibiting multiple drug resistance.The number of deaths caused by drug-resistant bacterial infections is increasing year by year,and new antibacterial agents are urgently needed to be developed.Bacteriophages and their lysing enzymes exhibit extraordinary potency in the development of new antibacterial drugs due to their high efficiency and specific bactericidal capabilities.Enterococcus faecalis(E.faecalis)is a Gram-positive bacterium,which is also known as streptococcus faecalis.E.faecalis is commonly found in the intestine,oral cavity,and reproductive tract of humans or other animals.Ectopic parasites can cause a variety of diseases such as endocarditis,meningitis,sepsis,urinary tract infections,and purulent abdominal infections.In recent years,the frequent appearance of multi-resistant E.faecalis has made antibacterial treatment more complicated,and therefore significantly threatening human health.In our previous work,a phage endolysin called Lys IME-EF1 was able to efficiently lyse E.faecalis,especially with nearly 30 strains of pathogenic E.faecalis causing clinically diverse diseases.It shows promising antibacterial application.The study found that the N-terminus of Lys IME-EF1 is a CHAP domain(cysteine,histidine-dependent amidohydrolases / peptidases domain),the middle is a Linker region,and the C-terminus is a cell-wall binding domain(CBD).In order to clarify the molecular mechanism of Lys IME-EF1 for efficient lysis of E.faecalis clinical strains,we determined its three-dimensional structure by X-ray crystal diffraction.Surprisingly,the endolysin Lys IME-EF1 shows a unique three-dimensional structure,and its overall structure is formed by a full-length Lys IME-EF1 and an additional three molecules of CBD to form a homotetramer.A ribosome binding site(RBS)was found upstream of the methionine codon(ATG)encoded by the 502-504 position in the ORF of the Lys IME-EF1 gene,which resulted in an additional three molecules of CBD in Lys IME-EF1 Causes.Subsequently,it was found that the unique heterotetrameric structure of Lys IEM-EF1 gave it the ability to lyse E.faecalis clinical strains,and destroying the Lys IME-EF1 CBD tetramer structure almost eliminated its ability to lyse E.faecalis clinical strains.Based on this,the key amino acids of Lys IME-EF1 CBD binding to the cell wall were found in this study by flowcytometry and cell lysis experiments.In summary,this study,for the first time,elucidates the mechanism of action of a single gene encoding a multi-component endolysin of E.faecalis from a structural and functional perspective.