The PTS EIIB Component Drives Strain-Specific Virulence in Listeria monocytogenes: Divergent Regulation of Biofilm Formation and Host Infection in High- and Low-Virulence Strains

<i>Listeria monocytogenes</i> (<i>L. monocytogenes</i>) is a Gram-positive intracellular pathogen capable of causing severe infections. The <i>Listeria</i> 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 <i>L. monocytogenes</i>, particularly modulating biofilm formation and host-pathogen interactions.