Two Nonadjacent Regions in Enteroaggregative Escherichia coli Flagellin Are Required for Activation of Toll-like Receptor 5

Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin. Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin. Most Gram-negative bacteria express flagella, surface structures that confer motility. Flagella are composed of a basal body that serves as a rotatory motor, a filament that extends into the space around the bacterium to provide motive force, and a hook that connects the two. The filament consists of a long homopolymer of a single protein, flagellin, with a small cap protein at the end. Polymerization of flagellin occurs as a result of relatively conserved structures at the N and C termini, although the intervening regions of the protein are highly diverse. The crystal structure of a central proteolytic fragment of flagellin was recently solved, contributing to an understanding of how these conserved structures are involved in filament formation (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google Scholar). In addition to their role in bacterial motility, an accumulation of recent evidence suggests that flagella also enhance the pathogenicity of certain organisms, either by promoting adherence to host tissues or by directly activating host inflammatory signaling pathways. Alterations in flagellar expression are associated with decreased virulence in several animal models of bacterial pathogenesis, includingPseudomonas aeruginosa lung infection (2DiMango E. Zar H.J. Bryan R. Prince A. J. Clin. Invest. 1995; 96: 2204-2210Google Scholar, 3Feldman M. Bryan R. Rajan S. Scheffler L. Brunnert S. Tang H. Prince A. Infect. Immun. 1998; 66: 43-51Google Scholar, 4Ramphal R. Arora S.K. Ritchings B.W. Am. J. Resp. Crit. Care Med. 1996; 154: S170-S174Google Scholar), Proteus mirabilis urinary tract adherence (5Mobley H.L. Belas R. Lockatell V. Chippendale G. Trifillis A.L. Johnson D.E. Warren J.W. Infect. Immun. 1996; 64: 5332-5340Google Scholar), Helicobacter pylori gastritis (6Ohta-Tada U. Takagi A. Koga Y. Kamiya S. Miwa T. Scand. J. Gastroenterol. 1997; 32: 455-459Google Scholar), and both nontyphoidal Salmonella enterica and S. typhi infections (7Wyant T.L. Tanner M.K. Sztein M.B. Infect. Immun. 1999; 67: 3619-3624Google Scholar, 8Wyant T.L. Tanner M.K. Sztein M.B. Infect. Immun. 1999; 67: 1338-1346Google Scholar, 9Ikeda, J. S., Schmitt, C. K., Weinstein, D. L., Metcalf, E. S., and O'Brien, A. D. (2000) 100th General Meeting of the American Society for Microbiology, Los Angeles, CA, May 21–25, 2000, pp. 81, Abstract B-175, American Society for Microbiology, Washington, D. C.Google Scholar, 10Igimi, S., Amano, F., and Kumagai, S. (2000) 100th General Meeting of the American Society for Microbiology, Los Angeles, CA, May 21–25, 2000, pp. 62, Abstract B-94, American Society for Microbiology, Washington, D. C.Google Scholar, 11Ciacci-Woolwine F. Blomfield I.C. Richardson S.H. Mizel S.B. Infect. Immun. 1998; 66: 1127-1134Google Scholar, 12Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Google Scholar). In addition, bacterial flagella from plant pathogens can induce immune responses in plants such as Arabidopsis thaliana and tomato (13Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Google Scholar, 14Bauer Z. Gomez-Gomez L. Boller T. Felix G. J. Biol. Chem. 2001; 276: 45669-45676Google Scholar, 15Meindl T. Boller T. Felix G. Plant Cell. 2000; 12: 1783-1794Google Scholar, 16Gomez-Gomez L. Felix G. Boller T. Plant J. 1999; 18: 277-284Google Scholar). These findings suggest that recognition of bacterial flagella by the innate immune system is a widespread phenomenon among higher eukaryotes. Recent reports indicate that the component of flagella responsible for eliciting host immune responses is the filament protein flagellin. Purified or recombinant flagellin causes interleukin (IL)-8, 1The abbreviations used are: IL, interleukin; TLR5, toll-like receptor 5; FliC, flagellin; EAEC, enteroaggregativeEscherichia coli ; LPS, lipopolysaccharide; PBS, phosphate-buffered saline. nitric oxide, and CCL20 release from Caco-2 intestinal epithelial cells (17Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Google Scholar, 18Sierro F. Dubois B. Coste A. Kaiserlian D. Kraehenbuhl J.P. Sirard J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13722-13727Google Scholar, 19Steiner T. Nataro J. Poteet-Smith C. Smith J. Guerrant R. J. Clin. Invest. 2000; 105Google Scholar), and intravenous flagellin causes a systemic inflammatory response in mice (12Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Google Scholar). Salmonella typhimurium flagellin is translocated across T84 cell monolayers during in vitro infection, where it acts basolaterally to cause IL-8 release (20Gewirtz A.T. Simon Jr., P. Schmitt C.K. Taylor L.J. Hagedorn C.H. O'Brien A.D. Neish A.S. Madara J.L. J. Clin. Invest. 2001; 107: 99-109Google Scholar). This effect was shown to be caused by activation of toll-like receptor 5 (TLR5) (21Gewirtz A.T. Navas T.A. Lyons S. Godowski P.J. Madara J.L. J. Immunol. 2001; 167: 1882-1885Google Scholar). Flagellin from Listeria monocytogenes also signals through TLR5, leading to systemic IL-6 release from challenged mice in a myeloid differentiation factor 88-dependent manner (22Hayashi F. Smith K.D. Ozinsky A. Hawn T.R., Yi, E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Google Scholar). Although it is evident from these reports that flagellin triggers immune responses in several systems, the mechanisms involved and the structural features of flagellin that elicit them remain unknown. In particular, it is unclear whether immune activation is specific to flagellins from particular microorganisms or conserved epitopes of flagellin form a pathogen-associated molecular pattern that is recognized by pattern-recognition receptors in a similar fashion to other pathogen-associated molecular patterns (such as lipopolysaccharide or CpG DNA). The studies described below were undertaken to answer these questions by examining the inflammatory structural elements of flagellin from enteroaggregative Escherichia coli strain 042, a well characterized diarrheal pathogen. We have previously shown that this flagellin (termed FliC-EAEC) potently elicits IL-8 secretion from several intestinal cell lines (19Steiner T. Nataro J. Poteet-Smith C. Smith J. Guerrant R. J. Clin. Invest. 2000; 105Google Scholar). Using both site-directed and random mutagenesis, we demonstrate that disruption of two specific regions in the constant domains of flagellin eliminates IL-8 induction and TLR5 activation. EAEC strain 042, originally isolated from a child in Chile, was obtained from James Nataro (Center for Vaccine Development, University of Maryland). The cloning and expression hosts Top10F′ and BL21 (DE3) pLysS were obtained from Invitrogen. To express recombinant flagellin,fliC genes were amplified from bacterial colonies by PCR, using the following primers: start, 5′-GGATCCATGGCACAAGTCATTAAT-3′; end, 5′-TTCGAATTAACCCTGCTGCAGAGA-3′. The resulting fragments were cloned into pCRT7/NT Topo TA (Invitrogen) according to the manufacturer's instructions. This plasmid contains an N-terminal His6 fusion under control of the T7 promoter. The vector containing the fliC gene from 042 (pfliC-EAEC) was used as the base for subsequent mutagenesis. Deletion mutants (with the exception of Del 0) were generated by two-step PCR and cloned into the BamHI and SfuI sites flanking the fliC gene in pfliC-EAEC. Point mutations were generated by either two-step PCR or by circular PCR withDpnI digestion to eliminate background wild-type plasmid. To create the Del 0 mutant, the BclI-StuI fragment corresponding to bases 34–176 of fliC-EAEC was excised, and the gel-purified plasmid backbone was blunt-ended with mung bean nuclease (MBI Fermentas, Burlington, ON, Canada) to maintain the open reading frame after religation. Transposon linker mutagenesis was performed on purified pfliC-EAEC using the EZ::TN in-frame linker insertion kit (Epicentre, Madison, WI). Kanamycin-resistant clones were screened for insertion within the fliC gene by restriction digestion. Clones of interest were digested with NotI and religated to yield plasmids with 19 amino acid insertions in-frame in all six frames. The exact site of the linker insertion was verified by sequencing. Expression of recombinant FliC from these clones was performed as described below; only clones yielding a product of the correct size seen by SDS-PAGE were analyzed further. pfliC-EAEC and its derived vectors with mutant fliC genes were maintained in Top10F′. For expression, plasmids were transformed into BL21(DE3) pLysSand grown in Luria broth with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml). Log-phase cultures were induced with 0.5 to 1.0 mm isopropyl-β-d-thiogalactopyranoside for 2 h at 37° C and then pelleted by centrifugation. Cells were lysed by two freeze-thaw cycles followed by and were by for were then for Caco-2 cell IL-8 release with or were purified to expression of a These recombinant flagellins were purified under by expression was verified by on after which were to and with a to in were used without to for on Caco-2 which not release IL-8 in response to or to For on flagellin was purified of by Flagellin this by not Caco-2 cells were obtained from the American and grown in with amino 2 mm and and were obtained from and and in or inflammatory For IL-8 release cells were into at 5 and used To flagellin for IL-8 were with of and protein to in were was and for IL-8 by ON, To the of to of flagellin were and the of the was used as the The and were by Felix were and purified by The was as a single and the was a purified were at mm or mm in and in containing the gene was obtained from A. Aderem of Washington, was used as a cells were obtained from B. of were maintained in with and cells were with and at in the was with without Cells were then with using 0.5 of plasmid and (MBI were for 5 at for and then with Expression of protein was at h by Cells were then with of and flagellin were were after h for IL-8 and cells were with and lysed by in of cell was by and was in a were by protein to a of to which IL-8 expression were were for h and for protein by the were to in and analyzed in on a were at of the from the below that were and analyzed using on that only a of E. coli flagellins were inflammatory (19Steiner T. Nataro J. Poteet-Smith C. Smith J. Guerrant R. J. Clin. Invest. 2000; 105Google Scholar), we that the of EAEC 042 flagellin be on epitopes in the variable domain of the To these six mutants of flagellin were shown in only of Del to release IL-8 from Caco-2 The in Del amino to of the of the N-terminal constant domain and the of the variable of the of the variable domain were required for as by the wild-type of Del Del Del and Del 5 with to of of in a mutants of with on Caco-2 in a To of the protein for random linker mutagenesis was performed on pfliC-EAEC. shown in and of mutants were of activity. all of these mutants were within a the of the constant This was of particular interest several that flagellar filament structure in the corresponding of S. typhimurium FliC (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google Scholar). of these and are analogous to and in the insertion sites of two of the linker mutations that eliminate To whether the of could be by filament several mutations were generated in this and These inflammatory that disruption by the acid linker insertion is that the or of and that filament structure the of inflammatory activity. of the mutants obtained were to elicit IL-8 release from Caco-2 with with wild-type These were purified and analyzed by shown in of these mutants (with the exception of the as although required higher to this insertion after a relatively its IL-8 release was that of in the this insertion is in the in mutant Del described of the mutants an insertion in the variable domain this of the protein is for Del which this is This suggests that structure or disruption of a specific is responsible for the of this linker with of of in a The potent of the mutants with its insertion in the at the C In addition, mutant with Del of the N-terminal of These regions of the protein were excised of the Salmonella flagellin (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google Scholar), their to FliC structure are not are to the of the flagellar with the by the mutants and (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google Scholar). This suggests that an interaction between these regions be required for flagellin as well as for inflammatory activity. has recently that several plant an immune response to flagellins from plant from the N of these flagellins (termed is a potent of this inflammatory which is by or with structural to toll-like contains a similar from through This the that signals inflammation through the corresponding and are shown in were in IL-8 from Caco-2 cells at as as were to IL-8 release caused by at a In addition, in this shown to or eliminate signaling in plants were in These mutations not the inflammatory of and the mutant Del described maintained inflammatory the of the containing the these findings demonstrate that the innate immune response to bacterial flagellin, although in both mammals and is at different of flagellins in the N-terminal conserved response was as in 14Bauer Z. Gomez-Gomez L. Boller T. Felix G. J. Biol. Chem. 2001; 276: 45669-45676Google L. Felix G. Boller T. Plant J. 1999; 18: 277-284Google IL-8 response in of to release IL-8 from Caco-2 cells was as described in to not with was in the plant flagellin containing this was not in this in mutants are shown in in a Plant response was as in 14Bauer Z. Gomez-Gomez L. Boller T. Felix G. J. Biol. Chem. 2001; 276: 45669-45676Google L. Felix G. Boller T. Plant J. 1999; 18: 277-284Google IL-8 response in of to release IL-8 from Caco-2 cells was as described in to not with was in the plant flagellin containing this was not in this in mutants are shown in from L. S. typhimurium in epithelial cells in a manner (21Gewirtz A.T. Navas T.A. Lyons S. Godowski P.J. Madara J.L. J. Immunol. 2001; 167: 1882-1885Google Scholar, F. Smith K.D. Ozinsky A. Hawn T.R., Yi, E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Google Scholar). was previously not whether also activates TLR5 and whether this activation is required for IL-8 The to this have to TLR5 in Caco-2 cells either by with a TLR5 or with a Caco-2 cells have a after differentiation in which is the at which flagellin IL-8 responses is cells were used as a and not release IL-8 in response to flagellin in their shown in cells with a TLR5 expression vector IL-8 in response to flagellin TLR5 expression not to IL-8 the flagellin mutants and Del not cause IL-8 release from cells These findings suggest that the features of flagellin which Caco-2 cells are also required to TLR5 in cells and that the of inflammatory of the flagellin mutants is caused by the to through for the of inflammatory of Del and could be of protein structure leading to as to the disruption of a on and Del was used to the of in and shown in the of and Del were with a at and at with the of S. typhimurium flagellin previously T. A. Nataro J. Guerrant R. J. Infect. 1998; Scholar). The mutant a of its to the were These findings suggest that the of inflammatory of these flagellins are caused by disruption of specific regions of the protein to major in protein The studies demonstrate the complexity of the innate immune recognition of flagellin. The results that large regions of the variable domain of can be or by linker insertion without inflammatory activity. linker mutagenesis identified only of the constant domain IL-8 activity. the of flagellin to linker insertions in this mutations including to filament effect on the inflammatory activity. of a of the N-terminal constant domain from this also a flagellin. In addition, linker insertions at a from these sites not eliminate inflammatory the results that epitopes of flagellin recognized by innate immune receptors in plants are not required for flagellin signaling in flagellins elicit potent inflammatory responses in a number of eukaryotic In at two systems, this inflammation is through TLR5 (21Gewirtz A.T. Navas T.A. Lyons S. Godowski P.J. Madara J.L. J. Immunol. 2001; 167: 1882-1885Google Scholar, F. Smith K.D. Ozinsky A. Hawn T.R., Yi, E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Google Scholar). receptors are characterized by to confer and to such as myeloid differentiation factor and of the identified are pathogen-associated molecular molecular in microorganisms not in eukaryotic hosts (such as LPS, and CpG DNA). TLR5 signaling is then flagellin a pathogen-associated molecular pattern that eukaryotic have to through with bacteria. For this to be the TLR5 recognition of flagellin have to be in the conserved domains of the protein, the variable domains are highly This in that flagellin from not Gram-negative bacteria be to TLR5, these conserved evidence in of this is that from E. coli were to induce IL-8 release from T84 cells and nitric release from cells (12Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Google Scholar, A.T. Navas T.A. Lyons S. Godowski P.J. Madara J.L. J. Immunol. 2001; 167: 1882-1885Google Scholar). are other reports of E. coli to induce the CCL20 F. Dubois B. Coste A. Kaiserlian D. Kraehenbuhl J.P. Sirard J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13722-13727Google Scholar), to TLR5 (22Hayashi F. Smith K.D. Ozinsky A. Hawn T.R., Yi, E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Google Scholar), or to elicit IL-8 release from epithelial cells T. A. Nataro J. Guerrant R. J. Infect. 1998; Scholar). the of whether inflammatory is a widespread of Gram-negative flagellins or a specific of pathogens to these questions is to flagellin mutants and to them for activity. are two reports of such with F. Mizel S.B. Infect. Immun. 2000; that release from was induced by Salmonella flagellin or by a the variable domain at Deletion of either the or of the variable domain the further an inflammatory a flagellin the variable domain was These findings to the that the variable domain of flagellin contains two inflammatory epitopes that structure by the constant domains is required to these epitopes In to these (17Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Google that a containing the and constant regions of Salmonella flagellin by an E. coli was as as wild-type flagellin. from these that the constant domains of flagellin the inflammatory epitopes and that the variable domains are only to the constant regions in of flagellin these questions to answer is that the protein at or in the of certain F. S. S. Namba K. J. Biol. Scholar), of the protein The that recombinant flagellin is as as flagellar from bacteria suggests that filament formation is not required for inflammatory activity. it is not whether of of recombinant occurs under the in In addition, certain bacteria can flagellin, and the importance of these to inflammation has not F. Blomfield I.C. Richardson S.H. Mizel S.B. Infect. Immun. 1998; 66: 1127-1134Google Scholar). The findings the molecular basis for innate immune recognition of flagellin. We demonstrate that linker insertions within a of the constant domain of both Caco-2 cell IL-8 release and responses in of of the N-terminal constant also the inflammatory response to flagellin. In contrast, of the variable domain to be for this activity. The of these mutations on the flagellin protein on the crystal structure of the S. typhimurium fragment (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google are shown in The linker insertions which eliminate inflammatory are the of the by the conserved domain of the The Del the by the N-terminal conserved These regions are by the crystal to form the of the flagellar The by the insertions is to the of the with of the N and C termini, which are to form were excised (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google Scholar, F.A. Imada K. Vonderviszt F. Y. Namba K. J. Biol. 2000; Scholar). This of the protein is the site of several in the Salmonella flagellin that confer filament structure (1Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Google Scholar). these findings suggest that innate immune recognition of flagellin a structural by the of the two long within the D1 The in inflammatory of mutants Del and could be by disruption of the of these leading to This model not the inflammatory of mutant which its insertion in the from the it have that flagellins by E. coli are these conserved structures to is that these flagellins or mutations or within the D1 be of within the D1 domain that innate immune recognition not filament studies to these questions on how flagellin is recognized by We and for and for of this We also Felix for the flagellin and for with

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