Flagellar hook protein FlgE initiates a proinflammatory response via mixed pathways (MPF2P.762)

Among components of bacterial flagellum, filament flagellin C mediates immunostimulation via TLR5 pathway. Here we showed that hook protein FlgE also participates in host-pathogen interactions and stimulates innate immunity. Recombinant P. aeruginosa FlgE was obtained via routine prokaryotic expression system. After treatment of immortalized human corneal epithelial cells (HCEC) with FlgE, microarray profiling revealed that up-regulated genes were highly enriched in inflammation/immunity-related pathways. Inclusion of anti-TLR5 antibodies or 130mM KCl in culture each partially decreased the effects of FlgE, suggesting a mixed signaling pathways involvement. Pull-down assay performed on FlgE with HCEC lysates and following Mass Spectrum assay of bound proteins suggested that caveolin, membrane type ATP Synthase, Rab5 subunits and prohibitin might couple with FlgE. In an intranasal delivery model in mice as well in an organoid culture of murine lung slices, FlgE induced upregulation of IL1b, IL6 and CXCL1 at both mRNA and protein levels as detected by real-time PCR or ELISA. When administrated with ovalbumin in mice, FlgE enhanced OVA-specific CD4 T cells expansion. 3D modeling of FlgE disclosed two segments that would participate in FlgE polymerization during hook formation, and altering these two sites decreased immunostimulatory potency of FlgE. In conclusion, FlgE bears immunostimulation activity when confronting host cells, and might be used as an immunomodulator/adjuvant.