title: Reconstitution of a functional Toll-like receptor 5 binding site in Campylobacter jejuni flagellin.
abstract: Bacterial flagellin is important for intestinal immune homeostasis. Flagellins from most species activate Toll-like receptor 5 (TLR5). The principal bacterial food-borne pathogen Campylobacter jejuni escapes TLR5 recognition, probably due to an alternate flagellin subunit structure. We investigated the molecular basis of TLR5 evasion by aiming to reconstitute TLR5 stimulating activity in live C. jejuni. Both native glycosylated C. jejuni flagellins (FlaA and FlaB) and recombinant proteins purified from Escherichia coli failed to activate NF-kappaB in HEK293 cells expressing TLR5. Introduction of multiple defined regions from Salmonella flagellin into C. jejuni FlaA via a recombinatorial approach revealed three regions critical for the activation of human and mouse TLR5, including a beta-hairpin structure not previously implicated in TLR5 recognition. Surprisingly, this domain was not required for the activation of chicken TLR5, indicating a selective requirement for the beta-hairpin in the recognition of mammalian TLR5. Expression of the active chimeric protein in C. jejuni resulted in secreted glycosylated flagellin that induced a potent TLR5 response. Overall, our results reveal a novel structural requirement for TLR5 recognition of bacterial flagellin and exclude flagellin glycosylation as an additional mechanism of bacterial evasion of the TLR5 response.
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, TD Utrecht, The Netherlands.
authors.forename: Marcel R
authors.initials: MR
authors.lastname: de Zoete
authors.forename: A Marijke
authors.initials: AM
authors.lastname: Keestra
authors.forename: Jaap A
authors.initials: JA
authors.lastname: Wagenaar
authors.forename: Jos P M
authors.initials: JP
authors.lastname: van Putten
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D002460:Cell Line; D002645:Chickens; D017931:DNA Primers / Q000235:genetics; D005408:Flagellin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012477:Salmonella enteritidis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051198:Toll-Like Receptor 5 / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D005408:Flagellin; C067138:flaA protein, bacteria; C416532:flaB flagellin