Exploring the molecular properties and bactericidal potential of Lysin B from mycobacteriophage WXIN.

The ever-increasing incidence of antimicrobial resistance has revived interest in phage-derived therapeutics against bacterial infections. Bacteriophage-encoded endolysins, which can destroy peptidoglycan of the host cell wall to release progeny, show potential as alternatives to traditional antibiotics. Owing to the highly complex cell wall of mycobacteria, most mycobacteriophages employ a two-component system, comprising a canonical endolysin (LysA) and a complementary enzyme, a specific mycolyl-arabinogalactan esterase referred to as Lysin B (LysB). In this study, LysB from mycobacteriophage WXIN demonstrated significant bactericidal activity against Mycobacterium smegmatis in the presence of Tween 80, with MIC/MBC values of 16/16 μg/mL. Its lipolytic activity was confirmed by the formation of clear precipitation zones on plates using the calcium precipitation assay, and its specific activity was estimated as 0.317 ± 0.021 U/mg by the pNPP hydrolysis assay. Site-directed mutagenesis of conserved residues in WXIN LysB revealed a typical catalytic triad (Ser96-Asp191-His268) from the α/β-hydrolase superfamily responsible for ester bond hydrolysis. Residue Gln97, adjacent to the reactive Ser96, was also critical for bactericidal and hydrolytic activities. Mutations in the conserved GNP motif were prone to form insoluble precipitates, which might reduce the structural stability of LysB. Additionally, the distinct morphological features and lipid composition of M. septicum, compared to M. smegmatis, likely account for its lack of susceptibility to the bactericidal activity of LysB, even in the presence of the surfactant Tween 80. Hence, LysB may have antimicrobial potential against mycobacterial infections, particularly when appropriate capsular-degrading drugs are explored as synergistic combinations.