Antibacterial Activity of a Fused Endolysin ENDO-1252/KL9P Against Multiple Serovars of Salmonella enterica

Abstract

Salmonella enterica (SE) is one of the most prevalent enteric pathogens globally and infects humans through contaminated food and water sources. The rising trend of antibiotic-resistant SE strains poses a critical threat to public health. Bacteriophage-encoded endolysins evolve a promising alternative as antimicrobial agents for combating SE infections. These enzymes target the peptidoglycan layer of bacterial cells, causing cell lysis and death. However, the use of endolysins against Gram-negative bacteria is challenging due to the composition of the outer membrane, which acts as a barrier preventing the endolysins from reaching the peptidoglycan layer. KL9P is a short amphipathic peptide containing both hydrophobic and hydrophilic regions, enabling it to interact with membranes and aqueous environments. In this study, an endolysin ENDO-1252, a Salmonella bacteriophage-encoded enzyme, was fused with a short peptide KL9P and produced an advanced endolysin, ENDO-1252/KL9P, which enhanced its ability to lyse multiple serovars of SE. ENDO-1252/KL9P exhibited potent lytic activity against SE strains with optimal bactericidal effects observed at 20 μM and incubation at 37°C in 20 mM HEPES buffer (pH 7.4). The lytic activity of this endolysin was also evaluated under various conditions, including pH ranges and temperatures, revealing that ENDO-1252/KL9P retained significant lytic activity across a range of temperatures (25°C–40°C) and pH values (6.0–9.0). The fusion protein demonstrated the highest lytic efficiency against SE serovars, specifically S. Enteritidis, S. Heidelberg, and S. Pullorum. Immunofluorescence analysis confirmed the binding of ENDO-1252/KL9P to the bacterial cell wall, indicating the co-localization with the peptidoglycan layer. These results suggest that ENDO-1252/KL9P is a promising antibacterial agent inhibiting predominant serovars of SE, showing enhanced lytic activity without outer membrane permeabilizers.