Eradication of MRSA biofilm-associated implant infections by low-immunogenic sustained-release lysostaphin fused to thermosensitive polypeptides

Abstract Staphylococcal bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA), are responsible for intractable infections—especially those associated with implants—through biofilm formation, presenting a worldwide menace. Antibacterial enzymes like lysostaphin (Lst) are promising for combating staphylococcal infections but limited by poor stability, high immunogenicity, and suboptimal pharmacokinetics, which hamper their clinical translation as antibiotic alternatives. Herein, we report a strategy of fusing thermosensitive elastin-like polypeptides (ELPs) to Lst to overcome these inherent drawbacks and eradicate MRSA biofilm-induced implant infections. Guided by AlphaFold2, we rationally engineered a chimeric Lst-ELP fusion protein that preserves potent lytic activity, demonstrates markedly enhanced stability, and exhibits substantially reduced immunogenicity relative to free Lst. The thermosensitivity of Lst-ELP enables in situ formation of a sustained-release depot upon subcutaneous injection, translating to an increased maximum tolerated dose, improved pharmacokinetics, and optimized biodistribution. Consequently, a single injection of Lst-ELP not only fully eradicated MRSA biofilms on implants and MRSA from the bloodstream and wound tissues, but also eliminated local and systemic inflammatory responses. This approach achieved complete clearance of MRSA biofilm-associated implant infections without adverse effects, highlighting its potential for clinical translation.