Designing the Next Generation of Endolysins: A Triple Strategy Integrating Bioinformatics Mining, Engineering...

The intensifying challenge of antimicrobial resistance (AMR) has outpaced traditional antibiotic discovery, leading the World Health Organization to call for the rapid exploration of alternative therapeutic options. Endolysins-phage-encoded peptidoglycan hydrolases-exhibit potent bacteriolytic activity against multidrug-resistant pathogens and show minimal propensity for resistance development. However, their clinical translation remains hindered by several critical barriers, including resource-intensive discovery processes, limited permeability through the outer membrane of Gram-negative bacteria, and various chemical, physical, and immunological challenges. In this review, we systematically summarize the fundamental characteristics of endolysins and integrate a tripartite strategy to enhance their therapeutic potential: bioinformatics-based pre-screening mining stage, mid-stage optimization stage of engineered modifications and post-enhancement stage combining encapsulation and formulation. Challenges inherent to each stage are critically analyzed, and future directions for endolysin development are discussed. Such multiple strategies provide a comprehensive roadmap for overcoming current barriers and accelerating the development of next-generation endolysin-based therapies against AMR.