Mining and engineering novel staphylococcal lysostaphins as species-specific antibacterial agents

ABSTRACT Antimicrobial resistance (AMR) presents a serious global healthcare challenge, with staphylococci, especially Staphylococcus aureus , being the main cause of deadly infections. Peptidoglycan hydrolases, particularly lysostaphins, are highly promising alternatives to traditional antibiotics, collectively termed enzybiotics, due to their specific lytic action against the bacterial cell wall. To thoroughly explore their therapeutic potential, we performed comprehensive genome mining of 118,127 staphylococcal and 500 closely related mammaliicoccal genomes. Ultimately, we discovered 19 unique lysostaphin sequences, divided into four distinct evolutionary clusters. By characterizing five novel lysostaphins alongside known references, we observed diverse lytic activities and species-specificities. Notably, we identified a novel lysostaphin variant from Staphylococcus simulans , Lys-Ssim, representing a previously unknown evolutionary cluster, which exhibits unmatched substrate promiscuity and an unprecedented lytic activity that extends beyond staphylococci. Additionally, structural modeling and site-directed mutagenesis revealed that peptidoglycan hydrolysis by these M23 metalloproteases depends on highly flexible conformations of the binding cleft. Using these structural insights, we successfully engineered lysostaphins with significantly improved enzyme selectivity for S. aureus while nearly eliminating activity against the skin commensal S. epidermidis . We also demonstrated the potential to engineer lysostaphins active against species other than staphylococci. Our findings show that natural staphylococcal lysostaphins are a rich, highly specialized resource for developing rationally engineered, microbiota-friendly antibacterial agents.