Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-kappa B and proinflammatory gene program activation in intestinal epithelial cells.
BACKGROUND
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-kappa B is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-kappa B during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-kappa B activation in all the cells; invasion of cells by the bacteria is not required to activate NF-kappa B.


RESULTS
Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and I kappa 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-kappa B 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-kappa B activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-kappa B 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.


CONCLUSIONS
These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-kappa B 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.
Deparment of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. tallant@ccf.org
Thomas
T
Tallant
Amitabha
A
Deb
Niladri
N
Kar
Joseph
J
Lupica
Michael J
MJ
de Veer
Joseph A
JA
DiDonato
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016543:Central Nervous System Neoplasms / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D004847:Epithelial Cells / Q000201:enzymology / Q000382:microbiology; D005408:Flagellin / Q000276:immunology* / Q000378:metabolism; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology; D019073:HT29 Cells / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase; D007249:Inflammation / Q000235:genetics; D007422:Intestines / Q000382:microbiology / Q000473:pathology; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D008562:Membrane Glycoproteins / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D011971:Receptors, Immunologic / Q000378:metabolism; D012475:Salmonella / Q000276:immunology; D012480:Salmonella Infections / Q000473:pathology; D015398:Signal Transduction / Q000502:physiology*; D012995:Solubility; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D001426:Bacterial Proteins; C507779:MYD88 protein, human; D008562:Membrane Glycoproteins; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D005408:Flagellin; D004247:DNA; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases