Evasion of Toll-like receptor 5 by flagellated bacteria Erica Andersen-Nissen*†, Kelly D. Smith*‡, Katie L. Strobe*, Sara L. Rassoulian Barrett§, Brad T. Cookson§¶, Susan M. Loganʈ, and Alan Aderem*,** *Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103; Departments of †Immunology, ‡Pathology, §Laboratory Medicine, and ¶Microbiology, University of Washington, 1959 Northeast Pacific Street, Seattle, WA 98195; and ʈInstitute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON, Canada K1A OR6 Edited by Ralph M. Steinman, The Rockefeller University, New York, NY, and approved April 26, 2005 (received for review March 11, 2005) Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved mucosal surfaces where they are in persistent contact with the site on bacterial flagellin that is required for flagellar filament epithelial cell barrier. assembly and motility. The ␣ and ␧ Proteobacteria, including the Several studies have demonstrated that bacterial flagellin is a important human pathogens Campylobacter jejuni, Helicobacter major stimulus of human epithelial cells (17–19) where it is recog- pylori, and Bartonella bacilliformis, require flagellar motility to nized by TLR5 (20). We demonstrate here that the ␧, as well as the efficiently infect mammalian hosts. In this study, we demonstrate ␣, Proteobacteria, although highly motile, are not recognized by that these bacteria make flagellin molecules that are not recog- TLR5. They possess specific changes in the TLR5 recognition site nized by TLR5. We map the site responsible for TLR5 evasion to on flagellin that destroy TLR5 recognition, and compensatory amino acids 89–96 of the N-terminal D1 domain, which is centrally amino acid changes in their flagellin molecules that preserve positioned within the previously defined TLR5 recognition site. motility. These changes are conserved among all flagellated mem- Salmonella flagellin is strongly recognized by TLR5, but mutating bers of the ␣ and ␧ Proteobacteria that infect mammals, suggesting residues 89–96 to the corresponding H. pylori flaA sequence that evasion of TLR5 may contribute to persistence of these abolishes TLR5 recognition and also destroys bacterial motility. To bacteria at mucosal surfaces. preserve bacterial motility, ␣ and ␧ Proteobacteria possess com- pensatory amino acid changes in other regions of the flagellin Materials and Methods molecule, and we engineer a mutant form of Salmonella flagellin Cell Lines and Bacterial Strains. CHO K1 cells (American Type that evades TLR5 but retains motility. These results suggest that Culture Collection) were grown as described (3). The following TLR5 evasion is critical for the survival of this subset of bacteria at bacteria were grown overnight, shaking in LB: Salmonella typhi- mucosal sites in animals and raise the intriguing possibility that murium strain TH4778 (FljBϪ͞FliCϩ; K. Hughes, University of flagellin receptors provided the selective force to drive the evolu- Washington, Seattle), Salmonella typhi (S. I. Miller, University of tion of these unique subclasses of bacterial flagellins. Washington, Seattle), Escherichia coli, clinical isolate H9049 (S. Swanzy, University of Washington, Seattle), Listeria monocytogenes flagellin ͉ innate immunity ͉ motility ͉ Helicobacter pylori ͉ Campylobacter strain 10403 (D. Portnoy, University of California, San Francisco), jejuni Legionella pneumophila, serogroup 1, Corby strain (K. Heuner, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany), Shigella flexneri (B. oll-like receptors (TLRs) are an important family of innate Cookson, University of Washington, Seattle), Pseudomonas aerugi- Timmune receptors that recognize pathogen-associated molec- nosa strain PAK (D. Speert, University of British Columbia, ular patterns, evolutionarily conserved structures that are required Vancouver, BC, Canada), Proteus mirabilis (S. Swanzy, University for microbial fitness and are not present in the host (1, 2). We have of Washington, Seattle), Bacillus subtilis (S. Swanzy, University of previously defined the amino acids on bacterial flagellin that are Washington, Seattle), and Staphylococcus aureus [American Type recognized by TLR5 (3). These amino acids are located in the highly Culture Collection (ATCC) 12599]. Serratia marcescens (clinical conserved D1 domain of the flagellin protein and cluster on the isolate, University of Washington, Seattle) was grown on LB agar convex surface that contacts adjacent flagellin monomers in the plates. Vibrio anguillarum strain 775 was grown at 15°C in tryptic soy flagellar protofilament. Mutating individual residues in the TLR5 broth (TSB) supplemented with 1.5% wt͞vol sodium chloride, and recognition site significantly reduced or completely abolished bac- terial motility, suggesting that evolving a functional flagellin that Edwardsiella tarda was cultured in TSB at 25°C (Maureen Purcell, evades TLR5 would require a complex series of mutations. University of Washington, Seattle). Bartonella bacilliformis (ATCC Recent reports conflict on the ability of TLR5 to recognize 35686) and Rhizobium (Ensifer) meliloti (ATCC 10310) were grown flagellin from a highly motile bacterium, Helicobacter pylori. Two according to ATCC recommendations. The following bacteria were grown under microaerophilic conditions according to ATCC rec- studies (4, 5), using HEK293 cell reconstitution systems, reported IMMUNOLOGY that H. pylori is recognized by TLR5. Two other groups (6, 7) ommendations: C. jejuni (ATCC 700819), H. pylori strain G27 (N. demonstrated that flagellin-responsive epithelial cell lines do not Salama, University of Washington, Seattle), H. pylori clinical iso- detect native or recombinant H. pylori flagellin, suggesting that H. lates (S. Swanzy, University of Washington, Seattle), Helicobacter pylori flagellin evades TLR5 recognition. hepaticus (ATCC 51449), and Helicobacter felis (ATCC 49179). W. H. pylori infects the gastric mucosa of approximately two- succinogens (ATCC 29543) was grown under anaerobic conditions thirds of the world’s population (www.cdc.gov͞ulcer͞md. according to ATCC recommendations. htm#howcommon) and is the primary cause of gastritis, peptic ulcer disease, gastric cancer, and mucosa-associated lymphatic NF-␬B Luciferase Reporter Assays. CHO K1 cells were transfected tissue (MALT) lymphomas (8). It belongs to the ␧ clade of the with human TLR5 cDNA cloned into the pEF6 V5͞His TOPO Proteobacteria, which includes commensals that inhabit the gut of vector (Invitrogen) and ELAM-LUC (Promega) plasmids, and ruminants (e.g., Wolinella succinogenes), and another extremely luciferase assays were performed as described (3). important human pathogen, Campylobacter jejuni (9, 10). C. jejuni infects the small and large intestine and is one of the most frequent causes of diarrhea worldwide (9). The ␧ Proteobacteria are flagel- This paper was submitted directly (Track II) to the PNAS office. lated, and their motility is necessary for efficient colonization and Abbreviation: TLR, Toll-like receptor. infection of the gut (10–16). The best studied organisms in this clade **To whom correspondence should be addressed. E-mail: [email protected]. (Helicobacter spp., Campylobacter spp., and Wolinella spp.) live on © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502040102 PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 ͉ 9247–9252 Downloaded by guest on October 3, 2021 Immunoblots. Flagellin from Bartonella bacilliformis was detected by 1B), despite vigorous motility (data not shown) and flagellin using a rabbit polyclonal antiserum to the protein (M. Minnick, expression (Fig. 1C). University of Montana, Missoula, MT). Flagellin from Rhizobium The data obtained with heat-killed bacteria (Fig. 1 A and B) were meliloti was detected by using a rabbit polyclonal antiserum (B. confirmed by using purified flagellin from nonstimulatory C. jejuni Scharf, Universita¨t Regensburg, Regensburg, Germany). Flagellin and H. pylori, as well as from stimulatory Salmonella typhimurium, from ␧ Proteobacteria was detected by using mouse monoclonal Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, anti-C. jejuni flagellin antibody (NovoCastra, Newcastle, U.K.). and Serratia marcescens (Fig. 1D). Purified flagellin from C. jejuni Flagellin from Salmonella typhimurium was detected by using rabbit and H. pylori did not stimulate human TLR5 over a wide concen- polyclonal anti-FliCi (Difco). Horseradish peroxidase conjugate tration range, whereas the other flagellins did (Fig. 1D). Because secondary antibodies (Zymed) were used for immunoblots. natural variants of LPS can act as antagonists (22), we determined whether nonstimulatory C. jejuni and H. pylori flagellins antagonize Purification of Native Bacterial Flagellin. Bacteria were grown as TLR5 activation by Salmonella typhimurium FliC. Incubation of described above and flagellin was purified as described (3). cells with 100-fold excess of either C. jejuni or H. pylori flagellin Protein concentration was determined by using the BCA assay failed to inhibit TLR5 activation by FliC, indicating that these (Pierce), and purity was confirmed by SDS͞PAGE and Coo- flagellins do not act as TLR5 antagonists (data not shown). massie blue staining. C. jejuni 81–176 flagellin was from S.M.L. We aligned the flagellin sequences of the bacteria tested with those from other flagellated bacteria, and constructed a molecular Flagellin Sequence Alignments. Flagellin sequences were aligned by tree (Fig. 1E). Flagellated