This study aimed to elucidate the functional characteristics of internalin InlJ from Listeria monocytogenes (Lm) in adhesion, invasion, intracellular proliferation, and mitochondrial damage and to clarify its regulatory mechanism in phage-mediated control of intracellular Lm infection. The results indicated that the ability of an inlJ deletion strain of Lm NJ05, NJ05-ΔinlJ, to adhere to and invade Caco-2 cells decreased by 56.37% and 17.74%, respectively, and the intracellular proliferation capacity also significantly decreased compared with the parental strain. The absence of InlJ resulted in decreased accumulation of mitochondrial reactive oxygen species (ROS) without affecting the membrane potential, thereby reducing the cytotoxicity of Lm. However, the NJ05-ΔinlJ strain displayed heightened sensitivity to phage vB-LmoM-SH3-3, with its efficiency of plating (EOP) 1.37 times that of the wild-type strain, and showed substantially enhanced lytic activity in vitro. Pre-treatment of Caco-2 cells with the phage led to a significant reduction in their adhesion to and invasion by Lm and its intracellular proliferation, and effectively alleviated mitochondrial damage caused by Lm. Thus, InlJ, a newly identified member of the internalin family, acts as an important factor influencing mitochondrial damage and regulates the interaction between Lm and phages, laying the foundation for the application of phages in combating intracellular bacterial infections.

In order to explore the role and function of the inlJ gene of Listeria monocytogenes (Lm) in phage sensitivity and biofilm, the inlJ gene-deficient strain Lm NJ05-ΔinlJ was constructed by homologous recombination. The growth, adhesion and invasion characteristics of the defective strain were identified. The results showed that compared with the wide-type strain Lm NJ05, the adhesion and invasion of RAW264.7 cells by Lm NJ05-ΔinlJ were significantly reduced to 20.05% and 4.42%, respectively. The efficiency of plaque formation was enhanced by 2.72 folds and phage vB-LmoM-NJ05 had a stronger lytic activity on Lm NJ05-ΔinlJ. Phage vB-LmoM-NJ05 at titers of 10⁵ and 10⁸ PFU/mL could completely inhibit and remove the biofilm of Lm NJ05-ΔinlJ, respectively. Transcriptional analysis of biofilm formation-related genes showed that the transcriptional levels of the degU, agrA, agrD, luxS, yneA, recA and hpt genes were significantly decreased to nearly zero in the defective strain after interacting with phage vB-LmoM-NJ05. In conclusion, deletion of the inlJ gene can enhance the phage sensitivity of Lm, and down-regulate the ability of cell invasion and biofilm formation. Therefore, the inlJ gene not only regulate Lm but also affect its interaction with phage, which lays a foundation for the development and application of phage biocontrol.