Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliC) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliC having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliC and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliC on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliC and its mutants, FliC and FliC, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliC have more complex interactions compared to the D1-D2 domain pair., and these domains also play a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliC has more missing side chain and characteristic amino acid peaks than FliC. The FliC group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliC and FliC groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliC affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.