Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.