Cationic Liposome-Fused Endolysin Lys40 Overcomes Outer Membrane Barriers and Enhances Survival in...

Multidrug-resistant (MDR) Salmonella enteritidis infections cause high mortality and devastating economic losses in poultry, pose severe threats to animal health and food safety, and create an urgent demand for effective antibiotic alternatives. Herein, we developed a cationic liposome-encapsulated bacteriophage endolysin Lys40 (designated Lys40-Lip), and systematically evaluated its therapeutic efficacy in a chick model challenged with Salmonella enteritidis strain S4. Recombinant Lys40 was encapsulated into cationic liposomes with an encapsulation efficiency (EE) of 34.83%. The resulting Lys40-Lip nanoparticles had a hydrodynamic diameter of 137.3 ± 4.1 nm, a high positive zeta potential of +42.5 ± 0.3 mV, and excellent stability, retaining 78.52% of its initial bactericidal activity after 56 days of storage at 4 °C. Following a three-day oral treatment in Salmonella enteritidis S4-infected chicks, Lys40-Lip significantly increased survival rates in a dose-dependent manner (72.22% to 88.89% for low-to-high dose vs. 44.44% in infected controls, p < 0.05) and reduced ileal Salmonella enteritidis S4 colonization by 28.8% compared to free Lys40. Histopathology revealed Lys40-Lip restored duodenal villus integrity and reduced jejunal and ileal inflammation. Serum cytokine analysis confirmed that Lys40-Lip effectively regulated the host inflammatory response, significantly downregulating the pro-inflammatory cytokines IL-1β and IL-6, and upregulating the anti-inflammatory cytokine IL-10. Crucially, liposomal encapsulation overcame the outer membrane barrier of Gram-negative bacteria via charge-driven fusion mediated by its high positive surface potential (+42.5 ± 0.3 mV), enabling targeted delivery of Lys40 without the need for EDTA or other outer membrane permeabilizers. Lys40-Lip significantly improved the therapeutic outcomes of avian salmonellosis via synergistic direct bactericidal activity, intestinal barrier protection and inflammatory response regulation, offering a promising nanotherapeutic strategy for the control of this disease in veterinary practice.