Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammat ory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). T o identify which domains induced proinflammatory activity, r6-histidine (6H IS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced I kappaB alpha loss poststimulation and NF-kappaB activation in Caco-2BBe c ells and was as potent as native-purified SD flagellin. IFN-gamma -primed D LD-1 cells stimulated with 1 mug/ml of 6HIS flag induced high levels of NO (60 +/- 0.95 muM) comparable to the combination of IL-1 beta and IFN-gamma (77 +/- 1.2) or purified native SD flag (66.3 +/- 0.98). Selected rSD flage llin proteins representing the D1, D2, or D3 domains alone or in combinatio n were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory med iators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl DI and D2 separated by an Escherichia coli hinge (ND1-2/ECH /CD2) indicated that D1 and D2 were bioactive when coupled to an ECH elemen t to allow protein folding. This chimera, but not the hinge alone, induced I kappaB alpha degradation, NF-kappaB activation, and NO and IL-8 productio n in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced hig h levels of TNF-alpha (900 pg/ml) in human monocytes comparable to native S D flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflamma tory activity of flagellin, therefore, resides in the highly conserved N an d C D1 and D2 regions.