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

Abstract

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliC_EcN) 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_EcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliC_EcN 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_EcN 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_EcN and its mutants, FliC_Δ174-506 and FliC_Δ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliC_EcN 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_Δ274-406 has more missing side chain and characteristic amino acid peaks than FliC_Δ174-506. The FliC_EcN group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliC_Δ174-506 and FliC_Δ274-406 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_EcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Keywords: Biological activity; Circular dichroism; Escherichia coli Nissle 1917; Surface-enhanced Raman spectroscopy; Truncated flagellin.