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Polycationic Peptide Engineering of Phage Endolysins Expands Host Range and Enhances Antibacterial and Antibiofilm Activities Against Bacillus Species
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DOI：10.1002/bit.70293.png)
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Abstract
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The exponential rise in antimicrobial resistance has highlighted the urgent need for the development of alternative antibacterial strategies, including phage-derived and engineered protein agents such as endolysins and artilysins. In this study, we report the rational design, expression, and functional characterization of novel artilysins derived from P19_358 lysin, a glycoside hydrolase family 24 enzyme. The native P19_358 lysin exhibits limited antibacterial activity, primarily against Bacillus subtilis, and requires EDTA pre-treatment to act against Gram-negative bacteria. To overcome this limitation, C-terminal fusion constructs were engineered using polycationic peptides, including a Cecropin A-derived peptide fragment and polycationic nonapeptide (PCNP), with and without flexible glycine-serine linkers. Structural and biophysical analyses confirmed that peptide fusion did not interfere with the native catalytic domain. Functional assays demonstrated that the engineered artilysins exhibited enhanced antibacterial activity and an expanded host range compared to the native enzyme. Among these constructs, Cecropin A fused artilysins (Art1 and Art2) showed the highest bactericidal activity, achieving up to ~5.4 log10 reductions in B. subtilis MTCC 121 T and ~4.9 log10 reductions in B. pumilus MTCC 1640 T and B. licheniformis MTCC 429 T. Additionally, Art2 exhibited pronounced antibiofilm activity, significantly reducing biofilm formation across the tested Bacillus species. Stability studies revealed that Art2 possessed superior thermal, pH, and salinity tolerance compared to the native lysin, retaining activity across a broad range of environmental conditions. These findings demonstrate that rational artilysin engineering can substantially enhance antibacterial potency, spectrum, and stability while preserving enzymatic function. Notably, Art2 emerges as a promising candidate for further development as a robust antimicrobial agent with potential applications in food safety and clinical settings.
Keywords:
artilysin
Bacillus
bacteriophage
Cecropin A
Endolysin
PCNP
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