Engineered phage-derived lysins effectively kill mycobacterial pathogens.

Antimicrobial resistance in pathogenic mycobacteria remains a critical challenge due to poor drug penetration through their complex cell wall, which necessitates prolonged multidrug regimens. Mycobacteriophages encode a lytic machinery that can disrupt this barrier. In this research article, we describe a modular mycolysin platform combining phage enzymes Lysin A and Lysin B with outer membrane-permeabilizing peptides and protein transduction domains using VersaTile shuffling technology. Screening the chimeric libraries against Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG), followed by the evaluation of selected mycolysin hits, identified potent candidates with minimum inhibitory concentration values as low as 1.28 μg/ml against M. bovis BCG and up to 75 μg/ml against pathogenic nontuberculous mycobacterium Mycobacterium avium. The three most potent mycolysins showed intracellular efficacy, serum stability, noncytotoxicity, in vivo proof-of-concept efficacy in rat wound and pulmonary infection models, and synergy with rifampicin treatment. This biotechnology framework illustrates the promise of translating phage enzymes into next-generation antimycobacterial therapies.