Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

Phage-derived peptidoglycan hydrolases (i.e. lysins) are considered promising alternatives to conventional antibiotics due to their direct peptidoglycan degradation activity and low risk of resistance development. The discovery of these enzymes is often hampered by the limited availability of phage genomes. Herein, we report a new strategy to mine active peptidoglycan hydrolases from bacterial proteomes by lysin-derived antimicrobial peptide-primed screening. As a proof-of-concept, five p eptidoglycan h ydrolases from the A cinetobacter b aumannii proteome (PHAb7-PHAb11) were identified using PlyF307 lysin-derived peptide as a template. Among them, PHAb10 and PHAb11 showed potent bactericidal activity against multiple pathogens even after treatment at 100°C for 1 hr, while the other three were thermosensitive. We solved the crystal structures of PHAb8, PHAb10, and PHAb11 and unveiled that hyper-thermostable PHAb10 underwent a unique folding-refolding thermodynamic scheme mediated by a dimer-monomer transition, while thermosensitive PHAb8 formed a monomer. Two mouse models of bacterial infection further demonstrated the safety and efficacy of PHAb10. In conclusion, our antimicrobial peptide-primed strategy provides new clues for the discovery of promising antimicrobial drugs. Bacteria are increasingly becoming resistant to antibiotics, leading to a rise in cases of dangerous diseases around the world. Innovative treatments are urgently needed, prompting researchers to turn to alternative approaches. One strategy is to focus on finding new ways to target the bacterial cell wall, a shield-like structure that wraps around the cell and protects it against the environment. Certain hydrolase enzymes can weaken this wall by breaking down its primary component, a type of molecules known as peptidoglycans. Lysins, for example, are produced by phage viruses (which prey on bacteria) and have gathered momentum as antimicrobial agents. However, clinical studies so far have largely focused on using peptidoglycan hydrolases against Gram-positive bacteria, in which peptidoglycans are directly exposed to the environment. In Gram-negative species, on the other hand, the cell wall includes an outer membrane that makes it harder for the enzymes to access their targets. Finding better phage-derived lysins that work against Gram-negative bacteria has been challenging so far, partly because this would require scanning the genomes of various phage species for candidates – an infor