Identification of Conserved Domains in Salmonella muenchen Flagellin That Are Essential for Its Ability to Activate TLR5 and to Induce an Inflammatory Response in Vitro

Abstract

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-κB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95–108 (motif N) in the N terminus and 441–449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface. The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-κB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95–108 (motif N) in the N terminus and 441–449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface. The whiplike flagellum of Gram-negative bacterium is implicated in microbial pathogenicity, serving as a means of propulsion and attachment and invasion of host epithelium. Recently it has been shown that the monomeric constituent of flagella, the 55-kDa protein flagellin, is a potent activator of pro-inflammatory eukaryotic cell signaling via its interaction with membrane-bound TLR5. Flagellin-TLR5 binding induces NF-κB nuclear translocation and activation of mitogen-activated protein kinase, resulting in the up-regulated expression of cytokines, such as tumor necrosis factor-α and IL 1The abbreviations used are: ILinterleukinNOnitric oxidePAMPSpathogen-associated molecular patternsTLRToll-like receptorsDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumIFN-γinterferon-γELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideDTTdithiothreitolEMSAelectrophoretic mobility shift assaysCHOChinese hamster ovaryLPSlipopolysaccharide.1The abbreviations used are: ILinterleukinNOnitric oxidePAMPSpathogen-associated molecular patternsTLRToll-like receptorsDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumIFN-γinterferon-γELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideDTTdithiothreitolEMSAelectrophoretic mobility shift assaysCHOChinese hamster ovaryLPSlipopolysaccharide.-6, chemokines, including IL-8, and pro-inflammatory free radical synthesizing enzymes, such as the inducible nitric-oxide synthase (1Feldman M. Bryan R. Rajan S. Scheffler L. Brunnert S. Tang H. Prince A. Infect. Immun. 1998; 66: 43-51Crossref PubMed Google Scholar, 2McDermott P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar, 3Ciacci-Woolwine F. McDermott P.F. Mizel S.B. Infect. Immun. 1999; 67: 5176-5185Crossref PubMed Google Scholar, 4Steiner T.S. Nataro J.P. Poteet-Smith C.E. Smith J.A. Guerrant R.L. J. Clin. Investig. 2000; 105: 1769-1777Crossref PubMed Scopus (218) Google Scholar, 5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, 6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). Because many Gram-negative pathogens express flagellin, it is conceivable that flagella contribute to the host inflammatory response to infection. Evidence in support of this role comes from experimental studies in which we have shown that administration of flagellin engenders systemic tissue injury, characterized by exudative lung inflammation, liver necrosis, and circulatory shock. Therefore, flagellin presents all the features of pathogen-associated molecular patterns (PAMPs) and can be regarded as another PAMP. interleukin nitric oxide pathogen-associated molecular patterns Toll-like receptors Dulbecco's modified Eagle's medium fetal bovine serum interferon-γ enzyme-linked immunosorbent assay green fluorescent protein phosphate-buffered saline phenylmethylsulfonyl fluoride dithiothreitol electrophoretic mobility shift assays Chinese hamster ovary lipopolysaccharide. interleukin nitric oxide pathogen-associated molecular patterns Toll-like receptors Dulbecco's modified Eagle's medium fetal bovine serum interferon-γ enzyme-linked immunosorbent assay green fluorescent protein phosphate-buffered saline phenylmethylsulfonyl fluoride dithiothreitol electrophoretic mobility shift assays Chinese hamster ovary lipopolysaccharide. Therapeutic opportunities to interrupt the pathologic effects of flagellin will be based upon a precise delineation of its interaction with TLR5 that induces pro-inflammatory signaling pathways. Biochemical analysis of Salmonella flagellin reveals that both conserved domains within both termini are important in inducing pro-inflammatory responses in cultured intestinal epithelial cells (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). Recent studies also suggest that the hypervariable domain is not involved in pro-inflammatory activation. A recombinantly expressed flagellin mutant, in which the central hypervariable domain was deleted, was shown not to detract from its ability to induce NF-κB signaling, suggesting that the highly conserved N and C termini are sufficient for TLR5 activation (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). Further definition of the pro-inflammatory flagellin sequences has been provided by evidence that neutralization of the N-terminal “RINSA” domain (amino acid 31–52) by monoclonal antibodies blocks the proinflammatory activity of recombinant flagellins of different bacterial origin (5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar). Recent studies (7Mizel S.B. West A.P. Hantgan R.R. J. Biol. Chem. 2003; 278: 23624-23629Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Jacchieri S.G. Torquato R. Brentani R.R. J. Bacteriol. 2003; 185: 4243-4247Crossref PubMed Scopus (60) Google Scholar) have also delineated the extramembranous region of TLR5 responsible for flagellin binding. The crystal structure of flagellin resembles an aircraft, with two wings and a central rod-shaped body. The core of the latter is derived from the α-helices of both the N and C termini, whereas the outer surface of the rod is constituted by the hypervariable domain (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). has been on Salmonella flagellin and its role in the of host pro-inflammatory recombinant flagellin or flagella from other Gram-negative such as and pro-inflammatory recombinant flagellin or flagella from Salmonella are the potent P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar, 3Ciacci-Woolwine F. McDermott P.F. Mizel S.B. Infect. Immun. 1999; 67: 5176-5185Crossref PubMed Google Scholar, 5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, F. A. J.P. S. A. 2001; PubMed Scopus Google Scholar). and flagellin induce pro-inflammatory flagellin is more potent and induces in L. Murthy Soriano F.G. Salzman A.L. Szabo C. Infect. Immun. PubMed Scopus Google Scholar). it was that pro-inflammatory activity of flagellin be to studies have the pro-inflammatory of flagellin. It is that Toll-like receptors are involved in the of and are an conserved of receptors that in of an domain with a or two and a region the receptor The of signaling is to that of the receptor both receptor receptor it has been shown that TLR5 signaling in cells F. Smith A. S. A. 2001; PubMed Scopus Google Scholar). of expression in the TLR5 a signaling that and F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, L. Mizel S.B. Infect. Immun. 2001; PubMed Scopus Google Scholar, S.B. Snipes J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Further studies also that TLR5 is expressed on the surface of epithelial and flagellin epithelial from the domain to the domain J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). a it was that TLR5 with as well as and that are involved in the of nitric-oxide synthase expression by flagellin S.B. Smith West A.P. J. Immunol. 2003; PubMed Scopus Google Scholar). The of the present was to further the regions in the N and C termini that contribute to the eukaryotic pro-inflammatory activity of flagellin. using deletion and we have the to a region in the and evidence is provided on the region of flagellin responsible for functional activation of and of Chinese hamster ovary cells in with and and cells in in Dulbecco's modified Eagle's medium with and and a of in and to to medium was the with the of flagellin in and cells with flagellin for The of and the of in the was by the as A. M. Szabo C. J. Google Scholar). epithelial cells an cell with the in in medium cells with recombinant flagellin for The medium was and for the of by of and of a bacterial expression S. muenchen sequences has been (5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar). an N-terminal of a and was used as the for and with used to flagellin was on of in a using and of for of for for and for with enzymes, and a the been with and and its deletion and the hypervariable two of and domains an expression in the flagellin by mutagenesis The recombinant by and in the of by sequence and of TLR5 was from a fetal lung A expression sequences to the N-terminal of TLR5 was by TLR5 sequences a eukaryotic expression of TLR5 protein in cells by by with and sequences by using a recombinant from of as the The with was with and the of the reporter of of the was in to an of and for with bacteria and with phosphate-buffered saline in and the by the was to a with binding and with binding The purified and protein by the The by and with to protein and cells and the with of using to the of medium was and cells with medium and to for a in and to in a medium and the medium was a for to and in medium the with the and in the of and The by for of and cell or nuclear as PubMed Scopus Google Scholar). cell cells from the by and in in and and on for the by and in nuclear cell in of A and of of was and the for by for and the in of and The and for protein by for and of the cell with of protein and on a a with bovine serum in and for and with or for and with The by binding assays using of NF-κB was from the of nuclear with binding and of and for in with as the was the the of the to and to antibodies and from to the as and for an the functional of the conserved regions of flagellin, both domains as and S. muenchen flagellin sequences in a used as a to sequences by with with enzymes, an expression with expressed in and purified to in to the of other bacterial and that with recombinant flagellin, by using as shown in various for activity by with for the of and of in was we recombinantly expressed with in the N terminus or C terminus or hypervariable region recombinant different a pro-inflammatory response in cells a of a response shown in recombinant flagellin either region to induce However, the recombinant the hypervariable domain a of with that of recombinant flagellin. we that the was in to the recombinant was also or in the activity of the protein sequences of to that of the flagellin hypervariable region the N and C domains not Taken together, results that the conserved N and C domains are important for pro-inflammatory signaling in eukaryotic cells and that the hypervariable in is not to this The of the hypervariable domain was this region in and sequence from to species. that both domains of flagellin are required to a pro-inflammatory we to the regions within domain that are required for pro-inflammatory of amino acids within the N terminus and or C terminus and recombinant for ability to as by the of in of flagellin proinflammatory in to the of the to a deletion of amino acids in the N terminus and and amino acids in the C terminus and A and Further in either the N terminus or the C terminus abolished of by flagellin. Taken together, results suggest that both termini of flagellin contribute to its pro-inflammatory activity and that involved in this are to a acid region in the N terminus (motif and a acid region in the C terminus (motif the highly conserved region of flagellins Gram-negative the region in the N was not for the of in activity of flagellin is by not also by the epithelial of pro-inflammatory cytokines, including tumor necrosis IL-8, and it has been shown that epithelial cells in response to flagellin T.S. Nataro J.P. Poteet-Smith C.E. Smith J.A. Guerrant R.L. J. Clin. Investig. 2000; 105: 1769-1777Crossref PubMed Scopus (218) Google Scholar, L. Szabo C. Murthy Virag L. A. Soriano F.G. Salzman A.L. 2003; PubMed Scopus Google Scholar, C.E. M. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. J.A. Infect. Immun. 2003; PubMed Scopus Google Scholar). The of eukaryotic pro-inflammatory expression in response to flagellin to cell intestinal epithelial cells are with lung epithelial which have been reported to expression in response to of flagellin as as L. Szabo C. Murthy Virag L. A. Soriano F.G. Salzman A.L. 2003; PubMed Scopus Google Scholar). and recombinant S. muenchen in with cells for the and release was by The results with cells in cells and that the N-terminal motif and the motif are both for proinflammatory regions in the conserved N and C termini also to expression in cells not for pro-inflammatory Because the the that within the N terminus and C terminus have a on the of pro-inflammatory signaling in eukaryotic we and a recombinant both of regions shown in in a of pro-inflammatory is either in the N terminus or C terminus have on flagellin We in an in which the hypervariable region was this we of the N terminus C terminus or both recombinant and in inducing in we recombinant two with in the N terminus and two with in the C terminus deletion of amino acids from the and a on pro-inflammatory whereas deletion from the C terminus and in a in pro-inflammatory results demonstrate that the N and C motifs are important for pro-inflammatory activity and suggest that the sequences in both domains in The results to further the of recombinant flagellins in which motif N and motif C the and the in pro-inflammatory activity of and may have from a of flagellin structure by deletion of a of conserved and this we in which all amino acids in motif N and motif C to different amino acids by The amino acid and sequences of and S. muenchen flagellin are shown in the and of motifs N and C to in the deletion of the the we the motif N or motif C or shown in or flagellin in motif C also reduced pro-inflammatory activity whereas deletion of the motif C abolished pro-inflammatory results suggest that flagellin two widely of which is to induce a proinflammatory with this or in motifs N and C abolished pro-inflammatory that sequences in both motifs are to an sequences in the N-terminal domain that are important for the pro-inflammatory activity of flagellin, we to the amino acids to induce a pro-inflammatory We in motif N acid was to or and was to two and based upon the crystal structure of flagellin, which reveals possible and and and of the and of S. flagellin, (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). The inducing activity of recombinant flagellins with is shown in the pro-inflammatory activity of all was to that of flagellin. Because NF-κB is to of many the nitric-oxide synthase we used electrophoretic mobility shift assays to flagellin with from cells with or flagellin and for NF-κB shown in the of flagellin or the a shift for whereas and activity to that of the flagellin or of NF-κB binding was reduced to a not with results of or with and activity in and However, with recombinant flagellin and NF-κB binding. the analysis antibodies the and the as of a well However, the of a monoclonal on the of the NF-κB support our that the pro-inflammatory activity of flagellin is in motifs N and C. the we evidence for possible of motifs N and C in the pro-inflammatory activity of flagellin. However, the not that motifs in flagellin the eukaryotic receptor for flagellin, TLR5. It is well that the pro-inflammatory activity of flagellin is via the TLR5 receptor (7Mizel S.B. West A.P. Hantgan R.R. J. Biol. Chem. 2003; 278: 23624-23629Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). cells TLR5 and a reporter have been to for by this TLR5 not to of the to such as cell or is by flagellin. binds to and the interaction induces NF-κB which is required for the of many pro-inflammatory F. Smith A. S. A. 2001; PubMed Scopus Google Scholar). a of studies have shown the importance of TLR5 in is the domains of flagellin and TLR5. We have the to functional activation of TLR5 by flagellin. We cell TLR5 protein and reporter the of an cells express of TLR5 expression of TLR5 in cells in NF-κB activity in response to with flagellin of cells to purified S. muenchen flagellin a of to the region of flagellin for its activation of we a of in and for the expression of in assays studies that the in and on NF-κB activation and However, further deletion of the N and C terminus sequences of flagellin and with and in which all the amino acids in motifs N and C are also to the reporter such that expression was with that of Taken together, results that the regions in flagellin required for pro-inflammatory activity and NF-κB activation are also required for its functional activation of the TLR5 is the monomeric protein of flagella, a as a means of propulsion or attachment to host is recently implicated as a of eukaryotic proinflammatory flagellin activates eukaryotic such as and intestinal and epithelial to release a of pro-inflammatory mediators in vitro and in including tumor necrosis as well as inducible P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar, 3Ciacci-Woolwine F. McDermott P.F. Mizel S.B. Infect. Immun. 1999; 67: 5176-5185Crossref PubMed Google Scholar, 4Steiner T.S. Nataro J.P. Poteet-Smith C.E. Smith J.A. Guerrant R.L. J. Clin. Investig. 2000; 105: 1769-1777Crossref PubMed Scopus (218) Google Scholar, 5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Immunol. PubMed Scopus Google Scholar, F. Mizel S.B. Infect. Immun. 1998; 66: PubMed Google Scholar). The of the present was to the regions and the amino acid within flagellin that contribute to its activation of pro-inflammatory was in response to from other that a central hypervariable domain is for the of pro-inflammatory activity P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar) or that pro-inflammatory activation was upon the highly conserved regions (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar, T.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The precise within the N and C termini that the pro-inflammatory host response to flagellin are this we have a analysis of recombinant flagellin in to that are for pro-inflammatory eukaryotic with the crystal structure of flagellin, our results a functional that a by which flagellin activates its host TLR5. of the structure of flagellin is based upon crystal structure analysis by (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). has reported that the N-terminal domain of flagellin two α-helices by a which is to our motif The in the N-terminal is the in the The domain in to the in the N-terminal domain and to α-helices are and the central whereas the hypervariable region the outer surface (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). upon our functional and the crystal structure from (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google we have a to for the of various on the structure of monomeric flagellin and to its functional activation of TLR5 regions of flagellin that are for activation of TLR5. we have a acid region within the N-terminal domain that a of amino acids in the N-terminal domain on proinflammatory activity by flagellin. However, deletion of motif N pro-inflammatory mutagenesis of all amino acids abolished pro-inflammatory of suggesting the of attachment within the motif are required for functional activation of TLR5. The of is by a S.G. Torquato R. Brentani R.R. J. Bacteriol. 2003; 185: 4243-4247Crossref PubMed Scopus (60) Google based on that that the amino acids of flagellin are involved in binding with amino acids of the TLR5 our binding sequences (motif N) with we have a acid region motif within the conserved C is for functional activation of TLR5. or of all amino acids in motif C reduced the pro-inflammatory not completely. we have sequences within the domains and C) that may be involved in and are for flagellin We that in either the N terminus or the C terminus have on pro-inflammatory activity by flagellin. However, pro-inflammatory Because are many of attachment the N and C which are in to our results suggest that in either the N or C termini not this and in both termini, are of the alignment of the two termini, resulting in a of pro-inflammatory the role of the hypervariable domain in the functional activation of flagellin has been a of in the this we have recombinant of flagellin in which the hypervariable domain has been deleted, the N and C termini We have in with a (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google that flagellin the N or C termini pro-inflammatory the the hypervariable domain in cells with that of flagellin results that the conserved N and C domains are required for pro-inflammatory and the hypervariable domain is and to the of pro-inflammatory expression (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). the flagellin the rod-shaped structure in our in the of the hypervariable Because the hypervariable region the N and C termini, it to that it the structure of flagellin, the the hypervariable pro-inflammatory to this we the activity of or in the We that and in recombinant pro-inflammatory activity the in flagellin. Taken together, results suggest that the hypervariable domain a role in a of the N and C a was not N-terminal in the that the C terminus may be more for the N The of various in the regions of flagellin on pro-inflammatory activation and is a and further will be required to the role of Because we have functional activation of TLR5 as a for the of flagellin our are we have not crystal of the various recombinant and can we that have the A to this is from electrophoretic studies we have that recombinant flagellins of on G. K. C. and A. L. and flagellin are in and amino acid flagellin by was and molecular of other flagellin in a further that the functional in pro-inflammatory activity involved in the which a in an Recent have our of the by which flagellin induces pro-inflammatory it has been that the TLR5 receptor bacterial flagellin derived from both and Gram-negative The interaction in the activation of which is required for the of many pro-inflammatory to be an of TLR5 for signaling (7Mizel S.B. West A.P. Hantgan R.R. J. Biol. Chem. 2003; 278: 23624-23629Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, S.B. Smith West A.P. J. Immunol. 2003; PubMed Scopus Google Scholar, H. S. A. J. 2003; PubMed Scopus Google Scholar, A. G. S. K. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, F. Smith A. J. Immunol. 2003; PubMed Scopus Google Scholar). in and cells not that all of the effects with various TLR5. we used cells with TLR5 and reporter the with flagellin that to induce a pro-inflammatory response in epithelial cells also to expression in our on the ability of the flagellin to induce or the ability of the to induce TLR5 activation. are with the that TLR5 is the receptor for and inflammatory Recent studies have the role of the in intestinal J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, C.E. M. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. T. K. T. A. H. S. H. M. T. H. H. J. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google in systemic and (5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, L. Murthy Soriano F.G. Salzman A.L. Szabo C. Infect. Immun. PubMed Scopus Google and in various of inflammation, and (1Feldman M. Bryan R. Rajan S. Scheffler L. Brunnert S. Tang H. Prince A. Infect. Immun. 1998; 66: 43-51Crossref PubMed Google Scholar, L. Szabo C. Murthy Virag L. A. Soriano F.G. Salzman A.L. 2003; PubMed Scopus Google Scholar). or flagellin may pro-inflammatory by and may and in of Gram-negative The of the proinflammatory activity of flagellin in the may studies in this