SFB flagellin mediates cell adhesion, endocytosis and immune regulation in germ-free mice

The results of <i>in vitro</i> co-culture experiments demonstrated significantly improved bacterial adhesion capabilities mediated by SFB flagellin. Germ-free mouse colonization assays revealed prolonged fecal persistence of flagellin-expressing strains. Immunohistochemical analysis of ileal tissues showed co-localization of recombinant bacteria with the lysosomal-associated membrane protein 2 (Lamp2), confirming cellular internalization. Furthermore, <i>mfliC3</i>-expressing <i>Escherichia coli</i> exhibited active invasion into MODE-K cells. RNA sequencing analysis identified significant enrichment of Th17 cell differentiation pathways in both ileum and hepatic tissues from Lac-<i>rfliC3</i>-colonized mice. Correspondingly, the Lac-<i>mfliC3</i> group showed elevated serum levels of Th17-associated cytokines including IFN-γ, IL-23p19, IL-17A, IL-5, and IL-6 compared to controls. Molecular docking simulations revealed high-affinity interactions between SFB flagellins and endocytic regulators endophilin A2 and αM integrin. These results demonstrate that SFB flagellin mediates bacterial-epithelial interactions through dual mechanisms of adhesion potentiation and active internalization, ultimately driving Th17-mediated immune responses.