The virulence factor LLO of Listeria monocytogenes can slow down biofilm formation and indirectly suppress phage lytic effect

Phage-based biocontrols are among the various tools available to control Listeria monocytogenes (Lm) in food and food processing environments. However, the effectiveness of phages against different Lm strains varies significantly. Prior research has demonstrated that the inactivation of virulence Listeriolysin O (LLO) leads to a loss of hemolytic activity and a reduction in overall virulence. In this study, we generated a hly gene-defective strain, NJ05-Δhly, which demonstrated a significant enhancement in motility despite the absence of hemolytic activity. Furthermore, NJ05-Δhly displayed diminished capacity for biofilm formation, which negatively correlated with glucose concentration. The efficiency of plating (EOP) for the defective strain decreased by approximately 100-fold following infection with Listeria phage vB-LmoM-NJ05, with a notable reduction in lytic effect observed at multiplicities of infection (MOI) of 1 or 10. With 10 ⁸ PFU/mL of phage treatment, the biofilm of Lm NJ05-Δhly was completely suppressed and removed. Moreover, phage treatment effectively decreased adhesion and invasion, as well as mitochondrial membrane damage, in addition to the accumulation of mitochondrial reactive oxygen species (ROS), even in the absence of LLO. Transcriptome analysis indicated that LLO is mainly associated with pathways involved in quorum sensing, purine metabolism, and peptidoglycan biosynthesis, among others. Notably, genes related to Lm phage sensitivity, pathogenicity, biofilm formation, and motility were significantly down-regulated. These findings suggest that LLO plays a crucial role in regulating Lm pathogenicity, phage susceptibility, and biofilm formation. This study provides evidence of the complex interplay between Listeria LLO and phage interactions during Lm infection.