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 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.

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