Directed evolution of the bacteriophage endolysin PlyC reveals a charge-mediated mechanism of thermal stabilization

Abstract

Bacteriophage endolysins are peptidoglycan hydrolases currently being clinically developed as antibacterial therapeutics. Despite possessing several advantageous antibacterial properties, many endolysins display only modest thermal stability. This characteristic hinders their therapeutic potential due to limited long-term stability and complex storage requirements. To overcome this limitation, an evolutionary-based protein engineering strategy was employed in a proof-of-concept study to increase the intrinsic stability of an endolysin. Using the multimeric endolysin PlyC as a model, directed evolution was applied to the thermolabile PlyCA catalytic subunit. After screening 18,000 mutants, the lead candidate identified was the point mutant PlyC (PlyCAN211H). The protonated PlyCAH211 side-chain stabilizes the subunit by forming favorable electrostatic field interactions with two acidic residues located in the N-terminal glycosyl hydrolase domain, possibly resulting in an extended linker structure being anchored to the surface of the domain. This mutation improved structural and thermal stability under conditions that maximize the stabilizing effect (pH 6.0) by 3.48 °C and 4.10 °C, respectively, and increased kinetic stability 18.6-fold over WT. Combining PlyCAN211H with a stabilizing mutation (PlyCAT406R) identified in an independent rational-based in silico screen of PlyC additively enhanced thermal stability by 7.46 °C at pH 6.0. Accordingly, with a thermal transition temperature of 54.60 °C, PlyC(PlyCAN211H,T406R) now represents a highly stable endolysin derived from a mesophilic phage. In addition to in silico screening, this validated directed evolution methodology can now be expanded to other endolysins and bacteriolytic enzymes for the purpose of increasing their thermal stability, thereby improving their practical utility as an antimicrobial biologic.