Cloning and expression analysis of fused holin-endolysin from RL bacteriophage; Exhibits broad activity against multi drug resistant pathogens

Abstract

Antibiotic resistance has become a major risk to community health over last few years because of antibiotics overuse around the globe and lack of new antibiotics development. Phages and their lytic enzymes are considered as an effective alternative of antibiotics to control drug resistant bacterial pathogens. Endolysins prove to be a promising class of antibacterials due to their specificity and less chances of resistance development in bacterial pathogens. Though large number of endolysins has been reported against gram positive bacteria, very few reported against gram negative bacteria due to the presence of outer membrane, which acts as physical barrier against endolysin attack to peptidoglycan. In the current study, we have expressed endolysin (RL_Lys) and holin fused at the N terminus of endolysin (RL_Hlys) from RL phage infecting multi drug resistant (MDR) Pseudomonas aeruginosa. Both endolysin variants were found active against wide range of MDR strains P. aeruginosa, Klebsella pneumonia, Salmonella Sp. and Methicillin Resistant Staphylococcus aureus (MRSA). Broth reduction assay showed that RL_Hlys is more active than RL_Lys due to presence of holin, which assist the endolysin access towards cell wall. The protein ligand docking and molecular dynamic simulation results showed that C- terminus region of endolysin play vital role in cell wall binding and even in the absence of holin, hydrolyze a broad range of gram negative bacterial pathogens. The significant activity of RL-Lys and RL_Hlys against a broad range of MDR gram negative and positive bacterial pathogens makes them good candidates for antibiotic alternatives.

Keywords: Endolysin; Holin; Molecular dynamic simulation; Multi drug resistant; Peptidoglycan; Protein-ligand docking.