Engineering the chimeric lysin Art-15 via NZ2114–PlySs2 fusion enhances strain-specific antibacterial activity

Abstract

The global rise of multidrug-resistant Gram-positive pathogens demands new antimicrobial strategies. Phage-derived endolysins such as PlySs2 show promising cell-wall targeting activity but may be limited by strain-dependent cell-envelope accessibility. We engineered Art-15, a chimeric lysin that fuses the lipid II-targeting peptide NZ2114 to PlySs2, to test whether enhanced targeting improves antibacterial activity. Art-15 was constructed using a (GGGGS)3 linker, expressed in E. coli, and purified by nickel affinity chromatography. Structural features were analyzed using AlphaFold2 and a transmembrane hidden Markov model. Antibacterial activity was evaluated through spot-on-lawn assays, minimum inhibitory concentration (MIC) determination, and five-hour OD600 turbidity assays. Art-15 was successfully expressed, with a recoverable soluble fraction corresponding to a ~36 kDa recombinant protein. Structural prediction indicated a compact fold without transmembrane helices. In spot assays, Art-15 showed a narrower activity spectrum than PlySs2 (12/51 vs. 27/51 isolates). However, against strain STA2505001, Art-15 exhibited a lower MIC (64 µg/mL) than PlySs2 or NZ2114 alone (128 µg/mL). In OD600 assays, Art-15 suppressed growth more effectively over five hours than either parental component.  Art-15 appears to trade broader-spectrum activity for increased strain-specific potency. The findings suggest NZ2114 may contribute to altered cell-envelope targeting and local exposure of the PlySs2 catalytic domain to peptidoglycan, although host-range narrowing indicates target accessibility remains strain-dependent.