Studies on the structure and stimulating effect on Caco-2 cells of flagellin and its truncated proteins of Escherichia coli Nissle 1917

Abstract

This study aimed to investigate the structural characteristics and their effects on stimulating Caco-2 cells of Escherichia coli Nissle 1917 flagellin (FliCEcN) and its truncated proteins, FliC△174−506 (D2-D3 domain deleted) and FliC△274−406 (D3 domain deleted). The experiment predicted the tertiary structure of FliCEcN by Alphofold2, analyzed the structural characteristics of FliCEcN, FliC△174−506 and FliC△274−406 by surface-enhanced Raman spectroscopy (SERS) and circular dichroism (CD), and detected the secretion levels of IL-6 (interleukin-6), IL-10 (interleukin-10) and TNF-α (tumor necrosis factor-α) after FliCEcN, FliC△174−506 and FliC△274−406 stimulated Caco-2 cells for 6 and 12 h, respectively. The results showed that the NH3-ends and COOH-ends of FliCEcN were highly conserved, mainly composed of α-helix; the middle domains were highly variable, mainly composed of β-sheet and random coil. The Raman peaks of FliC△174−506 and FliC△274−406 generally maintained the main chain peaks of FliCEcN, while the side chain and amino acid peaks were absent to varying degrees. The composition of the secondary structure of FliC△174−506 and FliC△274−406 was altered. FliCEcN, FliC△174−506 and FliC△274−406 stimulated Caco-2 cells to secrete cytokines IL-10, IL-6 and TNF-α differently. The complete FliCEcN structure could stimulate more secretion of IL-10; the FliC△174−506 group had higher secretion of IL-6; and the FliC△274−406 group had higher secretion of TNF-α. In conclusion, deletion of different domains of the hypervariable region of FliCEcN affects its SERS and CD spectrum and stimulates Caco-2 cells to secrete cytokines.