LIPI-4 as a Critical Modulator of InlB-Mediated Pathogenicity in Listeria monocytogenes

<i>Listeria monocytogenes</i> (Lm) is a foodborne pathogen whose virulence depends on the coordinated action of multiple virulence factors. Although deletion of either LIPI-4 or <i>inlB</i> reduces the virulence of Listeria monocytogenes, it remains unknown whether these two factors are functionally or regulatory connected. Therefore, we constructed an <i>inlB</i> deletion mutant and its complemented strain in the Lm928 and <i>ΔLIPI-4</i> 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 <i>inlB</i> expression in a cell-type-specific manner: <i>inlB</i> was downregulated in <i>ΔLIPI-4</i> under culture and HTR-8 infection, but upregulated during hCMEC/D3 infection-yet functional defects persisted in all cases. Biofilm assays showed that <i>ΔLIPI-4</i> and the double mutant exhibited enhanced biofilm formation, with the double mutant exceeding <i>ΔLIPI-4</i>, demonstrating synergistic enhancement. Motility assays indicated that LIPI-4 dominates bacterial movement, with <i>ΔLIPI-4</i> and the double mutant showing identical severe defects. Plaque formation analysis showed that LIPI-4 is essential for cell-to-cell spread, while <i>inlB</i> 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 <i>inlB</i> 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 <i>inlB</i> acting as a co-factor, whereas <i>inlB</i> unexpectedly promoted higher bacterial burdens in the brain, suggesting that <i>inlB</i> modulates LIPI-4-mediated neuroinvasion. Overall, our results establish LIPI-4 as the central determinant of Lm virulence, with <i>inlB</i> acting as a context-dependent co-factor that modulates LIPI-4-mediated pathogenesis in a cell type- and tissue-specific manner.