FlgE-TLR5 interaction and its association with immune and microbiota responses in Asian swamp eel fed Bacillus velezensis.

Bacillus velezensis has emerged as a promising probiotic in aquaculture; however, the mechanisms by which it modulates host innate immunity and gut homeostasis in teleost fish remain poorly understood. In this study, interactions among B. velezensis D1, TLR5-associated immune signaling, and gut microbiota dynamics were investigated using an integrated in vitro and in vivo model in Asian swamp eel. In vitro interaction assays demonstrated a selective interaction between eel TLR5 and the flagellar hook protein FlgE, which was validated by co-immunoprecipitation, showing a distinct band at approximately 48 kDa. No interaction was detected with FliC or FliD, and all negative controls were non-reactive, indicating selective association between FlgE and eel TLR5. This interaction suggests a potential link between TLR5-mediated probiotic sensing and downstream immune responses, which may be associated with subsequent intestinal remodeling and gut microbiota restructuring Dietary supplementation altered TLR5, IL-6, IL-1β, and TNF-α expression in spleen, liver, and gut, showing significant early upregulation (6-12 h, p < 0.01) and peak responses at 24-48 h (p < 0.001), with gene- and tissue-specific patterns. Histomorphological analysis revealed significant improvements in gut structure, including increased villus height and width, as well as intestinal wall and muscle layer thickness. Furthermore, 16S rRNA gene sequencing showed that probiotic supplementation significantly altered gut microbial communities. The probiotic group exhibited higher microbial richness, with 527 distinct OTUs compared to 239 in the control group. Beneficial phyla, including Firmicutes and Actinobacteria, increased, while Proteobacteria decreased. These findings suggest that TLR5-associated immune responses may be linked to the observed restructuring of gut microbiota, indicating a coordinated relationship between host immune activation and microbial community modulation following probiotic supplementation. Overall, this study provides insights into host-microbe interactions and supports the potential application of B. velezensis as a probiotic in sustainable aquaculture.