Structural And Function Study Of Enterococcus Faecalis Phage Endolysin Lys170

The abuse of antibiotics leads to different degrees of drug resistance of pathogenic bacteria in clinical practice.Enterococcus faecalis as a gram-positive bacterium,is a common pathogen that causes large-scale hospital infection,and its translocation in the host leads to diseases such as meningitis,sepsis and endocarditis.Because of macromolecular material has low permeability and thick cell walls and resistance genes and other factors,it can get clinical appeared the dung enterococcus resistance to different antibiotics,including vancomycin is considered in the treatment of gram-positive bacteria of the last line of defense,but also identify the vancomycin resistant strains,so seek new treatment methods become the urgent matter.Bacteriophages have become the natural enemies of bacteria,but their invasion of the host as a live virus has made development difficult.However,in the late stage of bacteriophage lysis,lysin is secreted,which can cleavage peptidoglycan,the main component of bacterial cell wall.Its essence is protein.Compared with bacteriophage,lysin has better controllability,higher specificity and stronger lytic ability,so it has become a research focus.The structure of phage lyase is generally composed of the N-terminal catalytic domain（CD）and the C-terminal cell binding domain（CBD）,which are connected by Linker.Lys170 from phage F170/08 is one of the earlier lyases discovered to lyse Enterococcus faecalis.However,the specific strain of lysis and the mechanism of lysis are unknown,so we solved this problem through biochemistry and structural biology methods.In this paper,the expression system of Escherichia coli was firstly used to express and purify Lys170,and then the clinically isolated pathogenic bacteria were screened to find that Enterococcus faecalis No.40 had high lytic ability.At the same time,X-ray crystalligraphy was used to analyze the protein structure with A resolution of 1.40 A.The final electron density map only had Lys170 CBD（amino acid 202-289）at the C-terminal,possibly due to the flexibility of Linker region.Structural analysis shows that Lys170 CBD exists in solution as tetramer,with each monomer consisting of three continuous β folds centered by an αhelix on each side.The mutation analysis of the seven amino acid sites bound by the tetramer showed that Y203 completely destroyed the tetramer interface of Lys170 CBD and lost the bactericidal ability,indicating that the formation of Lys170 CBD tetramer was crucial for the lysase to exert its lytic capacity.Considering Lys170 CBD binding to the cell wall,surface charge analysis of the structure resulted in mutation of six positively charged amino acid sites that might be involved in binding to the cell wall.Using plate titration,two sites R235 and R236 were identified as important for binding to the cell wall of Enterococcus faecium.In summary,this paper explores the structure and function of Lys170 to help understand its lysation mechanism and provide a theoretical basis for subsequent antimicrobial drug research and development.