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Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

Thomas Tallant1, Amitabha Deb12, Niladri Kar1, Joseph Lupica1, Michael J de Veer13 and Joseph A DiDonato1*

  • * Corresponding author: Joseph A DiDonato

  • † Equal contributors

Author Affiliations

1 Deparment of Cancer Biology, The Lerner Research Institute at the Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

2 Amitabha Deb, Massachusetts Biologics Labs, University of Massachusetts Medical School, 305 South Street, Jamaica Plain, MA 02130-3597, USA

3 Michael de Veer, Centre for Animal Biotech, Department of Veterinary Science, Melbourne University, Parkville, Victoria, 3010, Australia

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BMC Microbiology 2004, 4:33  doi:10.1186/1471-2180-4-33

Published: 23 August 2004



Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-κB is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-κB during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-κB activation in all the cells; invasion of cells by the bacteria is not required to activate NF-κB.


Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and Ikappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-κB via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-κB activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-κB activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both.


These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-κB and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition.