Listeria monocytogenes (L. monocytogenes) is a Gram-positive intracellular pathogen capable of causing severe infections. The Listeria pathogenicity island 4 (LIPI-4) encodes a phosphotransferase system (PTS) with its EIIB component playing a critical role in carbohydrate phosphorylation and virulence. However, the precise function of EIIB in virulence regulation across diverse pathogenic strains remains unclear. Here, we generated an EIIB deletion mutant (LM873ΔEIIB) and its complemented strain (CLM873ΔEIIB) from the low-virulence strain LM873, and performed comparative analyses with the high-virulence strain LM928 and its corresponding mutants. Deletion of EIIB differentially modulated biofilm formation: suppressing it in LM928 while enhancing it in LM873, accompanied by corresponding transcriptional changes in biofilm-associated and virulence genes. Both mutants exhibited impaired hemolytic activity, whereas motility attenuation was specific to LM928ΔEIIB. At the cellular level, LM873ΔEIIB enhanced adhesion to and invasion of Caco-2 but impaired intracellular proliferation in JEG-3; In contrast, LM928ΔEIIB promoted Caco-2 invasion while attenuating JEG-3 adhesion, invasion, and intracellular replication, as well as reducing invasion and proliferation in RAW264.7 macrophage. Animal experiments demonstrated that EIIB deletion attenuated LM928 colonization in the liver and spleen, but had no significant impact on LM873. Collectively, our findings establish EIIB as a strain-dependent virulence regulator in L. monocytogenes, particularly modulating biofilm formation and host–pathogen interactions.

Listeria monocytogenes (Lm) is a foodborne pathogen whose virulence depends on the coordinated action of multiple virulence factors. Although deletion of either LIPI-4 or inlB reduces the virulence of Listeria monocytogenes, it remains unknown whether these two factors are functionally or regulatory connected. Therefore, we constructed an inlB deletion mutant and its complemented strain in the Lm928 and ΔLIPI-4 backgrounds. We assessed bacterial growth, biofilm formation, motility, host cell interactions (adhesion, invasion, intracellular proliferation), plaque formation, mouse organ colonization. Growth curve analysis showed no significant differences among strains. qPCR revealed that LIPI-4 modulates inlB expression in a cell-type-specific manner: inlB was downregulated in ΔLIPI-4 under culture and HTR-8 infection, but upregulated during hCMEC/D3 infection—yet functional defects persisted in all cases. Biofilm assays showed that ΔLIPI-4 and the double mutant exhibited enhanced biofilm formation, with the double mutant exceeding ΔLIPI-4, demonstrating synergistic enhancement. Motility assays indicated that LIPI-4 dominates bacterial movement, with ΔLIPI-4 and the double mutant showing identical severe defects. Plaque formation analysis showed that LIPI-4 is essential for cell-to-cell spread, while inlB deletion unexpectedly enhanced plaque formation—an effect completely abolished in the absence of LIPI-4. Host cell assays across Caco-2, HTR-8, and hCMEC/D3 models revealed that LIPI-4 is the core determinant of adhesion, invasion, and intracellular proliferation, whereas inlB contributes in the context of LIPI-4 and its effects vary with the specific cellular process examined. In mice, LIPI-4 was essential for systemic colonization of the liver and spleen, with inlB acting as a co-factor, whereas inlB unexpectedly promoted higher bacterial burdens in the brain, suggesting that inlB modulates LIPI-4-mediated neuroinvasion. Overall, our results establish LIPI-4 as the central determinant of Lm virulence, with inlB acting as a context-dependent co-factor that modulates LIPI-4-mediated pathogenesis in a cell type- and tissue-specific manner.