Characterization and Engineering Studies of a New Endolysin from the Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity.

The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase (NALAA-2, EC 3.5.1.28) from bacteriophage PAC1 was characterized. The enzyme (Ami1) was cloned into a T7 expression vector and expressed in BL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity of Ami1 was confirmed using isolated peptidoglycan from . The antibacterial activity of Ami1 was investigated using live cells growing on agar plates. Two engineered variants of Ami1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes of bacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towards and the enterococci species and . The results of the present study suggest that Ami1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins.