Construction and Characterization of the Novel Engineered Endolysin ALC005: A New Alternative Antimicrobial Against Riemerella anatipestifer

The increasing serotypic diversity and multidrug resistance of Riemerella anatipestifer pose a serious threat to the poultry industry, necessitating novel antimicrobial agents. Although phage-derived endolysins offer rapid and specific bactericidal activity with low resistance potential, their efficacy against this Gram-negative pathogen is severely constrained by the outer membrane barrier, driving the need for strategies to enhance endolysin penetration. In this study, the native endolysin NA of phages vB_RanS_CRP2 was verified to lack antibacterial activity. To overcome this barrier, we constructed three chimeric proteins—ALC001, ALC005, and ALC007—by fusing a receptor-binding protein, a cell-penetrating peptide, or a polycationic nonapeptide, respectively. All three chimeras exhibited dose-dependent antibacterial activity, with ALC005 demonstrating the best performance. ALC005 achieved a lytic rate of 72.2% against the tested Riemerella anatipestifer strains, remained stable at 0–40 °C and pH 6–9, and was shown by transmission electron microscopy to exert its bactericidal effect by disrupting the bacterial cell envelope and inducing cell lysis. Collectively, cell-penetrating peptide fusion is a reliable and effective strategy to potentiate endolysin activity against Riemerella anatipestifer.