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-B 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 â€-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 â€-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.Flagellin, the monomeric subunit of the bacterial motility apparatus, is the natural ligand of the innate immune sensor Toll-like receptor 5 (TLR5) 3 (1). Activation of TLR5 by flagellin initiates a powerful host response that provides crucial signals for maintaining intestinal immune homeostasis (2, 3). The immunostimulatory properties make flagellin an attractive vaccine carrier protein and potent vaccine adjuvant. Its intrinsic adjuvant activity is currently being employed in experimental recombinant vaccines against human influenza, West Nile fever, malaria, tuberculosis, and plague (4 -9). In addition, flagellin-induced immune activation protects the intestine and other tissues against lethal irradiation due to potent TLR5-mediated anti-apoptotic effects (10, 11).The immunological impact of flagellin stimulation has driven several bacterial pathogens to evolve mechanisms to escape the effective TLR5-mediated host defense. In Salmonella enterica serotype Typhi, this is achieved by repression of flagellin expression and secretion (12), whereas Listeria shuts off flagellin expression at the host temperature of 37°C (13). The flagellins of the âŁ-and â-proteobacteria, which include the major food-borne pathogen Campylobacter jejuni and the gastric pathogen Helicobacter pylori, fail to activate TLR5 altogether (14 -18). For these organisms the consequences of TLR5 evasion for infection are currently unknown. It has been demonstrated that purified H. pylori flagellin induces severely impaired adaptive immune responses in comparison to TLR5-activating flagellins (19).The flagellin protein of C. jejuni clearly differs from bacterial flagellins that do activate TLR5. Electro...