Simultaneous enzyme grafting on bio-inspired scaffolds for antibacterial protection

Abstract

Surface bacterial contamination represents a crucial health and industrial concern which requires new strategies to be continuously developed. Successful antibacterial surfaces are characterized by a combination of durable and broad-spectrum antimicrobial actions. Herein, we present a bio-inspired strategy mimicking natural cellulosome to simultaneously immobilize multiple enzymes with antibacterial activity onto surfaces. The grafting strategy leverages the strong biomolecular interaction between receptors on a scaffold protein anchored on the substrate and ligands added to the enzymes. As a proof of concept, lysozyme and lysostaphin were chosen to target the bacterial cell wall, and DNase I to degrade DNA released during cell lysis, known to promote bacterial adhesion which can later lead to biofilm formation. The specificity of the ligand/receptor interaction was confirmed by biochemical and AFM-based single-molecule force spectroscopy assays, thus demonstrating successful co-immobilization of the three enzymes on the protein scaffold. Then, the antibacterial protection was evaluated against Staphylococcus aureus, Escherichia coli and Micrococcus luteus by viability tests which revealed long-term antimicrobial protection of the multi-enzymatic scaffold on both Gram-positive and Gram-negative bacteria. After 24 hours of contact, the system induced lysis of 71 to 85% of bacteria, and its antimicrobial properties remained effective after 5 days even with several cumulative waves of bacterial contamination. This work demonstrates the relevance of bio-inspired multi-enzymatic scaffolds for antibacterial protection, providing long-term and broad-spectrum action.