Adjuvant Effects of Deleting Hypervariable Domains of FliC of Escherichia coli Nissle 1917

Abstract

Bacterial flagellin (FliC) can be used as a TLR5 ligand-like adjuvant. However, the sequences of hypervariable regions(HVR) of FliC from different bacteria vary, and their effects on adjuvants remain unclear. In this study, FliC_Δ274–406 (deleting of D3 domain) and FliC_Δ174–506 (deleting of D2-D3 domain) from Escherichia coli Nissle 1917 FliC (FliC_EcN) were constructed and expressed in host bacteria, BL21. Purification was conducted using affinity chromatography on a Ni-NTA column, validation was done using SDS-PAGE and western blotting, the antigenicity and immunogenicity were detected using ELISA, and adjuvant effects were evaluated in Caco-2 cells and mice. The results showed that FliC_EcN was mainly expressed in the bacterial supernatant, and the two truncated flagellins were expressed as inclusion bodies. Compared with FliC_EcN, both FliC_Δ274–406 and FliC_Δ174–506 had considerable decreased antigenicity and immunogenicity. In Caco-2 cells, FliC_Δ174–506 had a higher ability to promote the secretion of IL-8 than FliC_EcN and FliC_Δ274–406. In mice, FliC_Δ174–506 showed a comparable adjuvant level to FliC_EcN, while FliC_Δ274–406 was less effective. Our data shows that adjuvant effects of FliC_EcN with deletion of different regions of its HVR are inconsistent, and deleting its entire D2–D3 domain is better.