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Evasion of Toll-Like Receptor 5 by Flagellated Bacteria

Evasion of Toll-Like Receptor 5 by Flagellated Bacteria

Evasion of Toll-like receptor 5 by flagellated

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 barrier. assembly and motility. The ␣ and ␧ , including the Several studies have demonstrated that bacterial flagellin is a important human jejuni, major stimulus of human epithelial cells (17–19) where it is recog- pylori, and 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 , which is centrally changes in their flagellin molecules that preserve positioned within the previously defined TLR5 recognition site. motility. These changes are conserved among all flagellated mem- 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 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), coli, clinical isolate H9049 (S. Swanzy, University of Washington, Seattle), Listeria monocytogenes flagellin ͉ innate immunity ͉ motility ͉ ͉ Campylobacter strain 10403 (D. Portnoy, University of California, San Francisco), jejuni pneumophila, serogroup 1, Corby strain (K. Heuner, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany), flexneri (B. oll-like receptors (TLRs) are an important family of innate Cookson, University of Washington, Seattle), aerugi- Timmune receptors that recognize -associated molec- nosa strain PAK (D. Speert, University of British Columbia, ular patterns, evolutionarily conserved structures that are required Vancouver, BC, Canada), 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]. 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. 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 tarda was cultured in TSB at 25°C (Maureen Purcell, evades TLR5 would require a complex series of mutations. University of Washington, Seattle). (ATCC Recent reports conflict on the ability of TLR5 to recognize 35686) and (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 (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, , 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., succinogenes), and another extremely luciferase assays were performed as described (3). important human pathogen, (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 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 , , Listeria monocytogenes, anti-C. jejuni flagellin antibody (NovoCastra, Newcastle, U.K.). and (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 bacteria that were recognized by TLR5 using CLUSTALW (www.ch.embnet.org͞software͞ClustalW.html) were spread among many branches of the tree and were members and displayed with BOXSHADE (www.ch.embnet.org͞software͞ of either the Proteobacteria (␤ and ␥ clades) or of the Spirochetes BOX࿝form.html). A molecular tree was displayed with and . Flagellated bacteria that were not recognized by PHYLODENDRON. TLR5 clustered in only two clades, the ␣ and ␧ Proteobacteria.

Creation of Flagellin Chimeras. Salmonella typhimurium fliC (Gen- The N-Terminal D0-D1 Domain of Flagellin Is Required but Not Suffi- Bank accession no. D13689) and H. pylori 26695 flaA (GenBank cient for TLR5 Recognition. The most likely explanation for our accession no. NC࿝000915) were amplified and cloned into the NcoI observations was that ␣ and ␧ Proteobacteria possess specific amino and XbaI sites of the ptrc99a plasmid containing an N-terminal acid changes that prevent TLR5 recognition. The crystal structure 6xHIS tag. Flagellin D0-D1 domain chimeras and 89–96 aa sub- of flagellin from Salmonella typhimurium (FliC) has been deter- stitutions were made by using a standard PCR mutagenesis strategy mined (23, 24) and is shown in Fig. 2A. FliC is divided into four (21). All mutations were verified by DNA sequencing. major domains, D0, D1, D2, and D3, comprised of regions from both the N- and C-terminal ends of the protein. FlaA, flagellin from Purification of Flagellin Chimeras. Escherichia coli BL-21RIL cells H. pylori, has not been crystallized, but its amino acid sequence is (Stratagene) were transformed with the flagellin chimera plasmids, highly similar to FliC in the D0-D1 domains. Fig. 2B shows a linear and flagellin expression was induced by 3–4 h of culture in the representation of both molecules. presence of 1 mM isopropyl ␤-D-thiogalactoside (IPTG). The We constructed a panel of flagellin chimeras by combining 6xHIS FliC was purified under native conditions by using B-PER domains from stimulatory Salmonella typhimurium FliC and non- protein extraction reagent (Pierce) and a TALON metal affinity stimulatory H. pylori FlaA; in particular, we wanted to determine column (BD Biosciences). The 6xHIS FlaA and all chimeras were what portions of FliC would convert FlaA into a TLR5 agonist. We purified from inclusion bodies under denaturing conditions and expressed and purified the chimeric proteins from flagellin- were then refolded. Insoluble proteins were spun out of the solution deficient Escherichia coli and demonstrated their purity by using at 16,000 ϫ g in a microcentrifuge for 10 min. Flagellins were heated SDS͞PAGE (Fig. 2C). A chimera containing both the N- and to 70°C for 20 min to depolymerize any filaments into flagellin C-terminal D0-D1 domains of FliC with the D2-D3 domain of FlaA monomers. Protein concentrations were determined by using the activated human TLR5 at higher doses than wild-type FliC (EC50 BCA assay (Pierce), and purity was confirmed by SDS͞PAGE and 160 ng͞ml) (Fig. 2 B and D), in agreement with another published Coomassie blue staining. study (25). The converse, a chimera containing the N- and C- terminal DO-D1 domains of FlaA with the D2-D3 domain of FliC Motility Assays. Salmonella typhimurium BC696 [SL1344 was inactive (Fig. 2 B and D). Molecules containing only the fliCϪfljBϪ, (3)], transformed with flagellin constructs, were stab- N-terminal or C-terminal D0-D1 domain of FliC were also inactive inoculated into motility plates [LB media containing 0.3% agar, (Fig. 2 B and D). A FliC molecule containing the C-terminal D0-D1 with 50 ␮g͞ml ampicillin and 1 mM isopropyl ␤-D-thiogalactoside domain of FlaA retained weak agonist activity, whereas a FliC (IPTG) for flagellin constructs]. Cultures were incubated upright at molecule containing the N-terminal D0-D1 domain of FlaA was 37°Cfor8h(Salmonella typhimurium) or at 26°C for 5 days completely inactive (Fig. 2 B and D). The data demonstrate that (Rhizobium meliloti), and then photographed. TLR5 agonist activity is contributed by both the N- and C-terminal D0-D1 domains of FliC; however, the N-terminal D0-D1 domain of Results FliC is more critical for this activity. Flagellins from ␣ and ␧ Proteobacteria Are Not Detected by TLR5. We screened flagellated bacteria for their ability to be recognized by Definition of Amino Acids Within the N-Terminal D0-D1 Domain of human TLR5. As predicted, a wide variety of heat-killed flagellated Flagellin Required for TLR5 Activation. Having demonstrated the bacteria, including Salmonella typhimurium, Salmonella typhi, Shi- importance of the N-terminal D0-D1 domain of flagellin in TLR5 gella flexneri, Pseudomonas aeruginosa, Listeria monocytogenes, Ser- recognition, we wanted to define the specific amino acids respon- ratia marcescens, , and Escherichia coli stim- sible for TLR5 activation in the ␤ and ␥ Proteobacteria and to ulated TLR5-dependent NF␬B activation (Fig. 1A). Similar results determine whether the loss of these amino acids was responsible for were obtained with , Bacillus subtilis, Edwardsiella the lack of activity of flagellins from the ␣ and ␧ Proteobacteria.We tarda, and V. anguillarum (data not shown). As expected, nonflagel- compared the flagellin N-terminal D0-D1 domain amino acid lated Staphylococcus aureus did not trigger TLR5 (Fig. 1A). Inter- sequences from TLR5-stimulatory bacteria to bacteria that do not estingly, Bartonella bacilliformis, Rhizobium (Ensifer) meliloti, C. activate TLR5. Interestingly, the highest proportion of amino acid jejuni, H. pylori (strain 26695 and two clinical isolates), H. hepaticus, differences between TLR5-stimulatory and nonstimulatory bacte- H. felis, and W. succinogenes were not recognized by TLR5 (Fig. ria was within a specific region of the D1 domain (amino acids

9248 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502040102 Andersen-Nissen et al. Downloaded by guest on October 3, 2021 Fig. 1. Flagellin from ␣ and ␧ Proteobacteria is not detected by TLR5. (A and B) Approximately 1 ϫ 105 CHO cells stably expressing human TLR5 and an NF-␬B luciferase reporter were stimulated for 4 h with heat-killed samples of stationary phase bacterial cultures diluted 1:100 in media. Data represent % fold induction of luciferase activity relative to maximal stimulation achieved with Salmonella typhimurium (Ϸ10- to 12-fold increase in luciferase activity over stimulation with LB alone) for at least three independent experiments, each run in triplicate. Error bars represent 1 SD. Control CHO cells stably transfected with empty expression vector and NF-␬B luciferase reporter did not respond to flagellated bacteria (data not shown). (C) Immunoblots of sonicated samples of stationary phase bacterial cultures diluted 1:10. (C Left) Bartonella bacilliformis flagellin, as detected with anti-flagellin antiserum. (Center) Rhizobium (Ensifer) meliloti flagellin, as detected with anti-flagellin antiserum (bands at Ϸ23 and 26 kDa likely represent degraded flagellin). (Right) ␧ Proteobacteria flagellins as detected with anti-C. jejuni flagellin antiserum (additional band in H. pylori lanes likely represents crossreacting hook protein Ϸ75 kDa). The left margins show molecular size in kDa. (D) TLR5 dose-response curve to purified flagellins. Flagella were sheared from bacteria and purified by ultracentrifugation, and purity was confirmed by SDS͞PAGE and Coomassie blue staining. Purified flagellin was incubated with CHO-hTLR5 cells for 4 h. ST, Salmonella typhimurium; EC, Escherichia coli; PA, Pseudomonas aeruginosa; LM, Listeria monocytogenes; HP, H. pylori; CJ, C. jejuni. Error bars represent 1 SD. (E) Molecular tree of flagellin sequences from flagellated bacteria, constructed by using CLUSTALW and displayed with PHYLODENDRON. Ϫ , flagellated bacteria tested that did not activate TLR5; *, flagellated bacteria tested that activated TLR5. IMMUNOLOGY

89–96) (Fig. 3A). Furthermore, this block contained three of the contact between monomers is mediated by the association of thirteen amino acids in FliC that we had previously defined as complementary convex (green and yellow) and concave (dark blue) important for TLR5 activation (residues 89, 90, and 94) (3). These surfaces (Fig. 4A Left) (23). The amino acids required for TLR5 amino acids were highly conserved within all bacteria that activate recognition are found within the convex surface, explaining their TLR5, but were different in the ␣ and ␧ Proteobacteria (Fig. 3A). crucial contribution to filament assembly and motility (3). Replacement of amino acids 89–96 of FliC with the corresponding amino acids from FlaA abolished the TLR5-agonist activity of FliC, Compensatory Mutations in ␧ Proteobacteria Flagellin Preserve Bac- emphasizing the importance of these amino acids (Fig. 3 B and C). terial Motility. In contrast to the ␤ and ␥ Proteobacteria, the ␣ and The question arises as to why this stretch of 8 aa that are crucial ␧ Proteobacteria are not recognized by TLR5, yet are motile. We for TLR5 recognition of flagellin has remained highly conserved hypothesized that preservation of motility was due to compensatory among ␤ and ␥ Proteobacteria, because their mutation would allow amino acid changes within the contact surfaces between flagellin pathogen evasion of host surveillance. The answer lies in the monomers that retain the ability to form filaments that propel the observation that these 8 aa are also crucial for flagellar filament bacterium, yet destroy TLR5 recognition. formation (data not shown) and motility (Fig. 3 B and D). Crys- To test this hypothesis, we chose a simple model system using a tallographic analysis of flagellar filaments has demonstrated that single amino acid mutation in FliC from Salmonella typhimurium

Andersen-Nissen et al. PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 ͉ 9249 Downloaded by guest on October 3, 2021 Fig. 2. The N-terminal D0-D1 domain of flagellin is required but not sufficient for TLR5 recognition. (A) Structure of FliC from Salmonella typhimurium with major do- mains labeled. The switch region (S) con- nects D0 and D1, but is not labeled. The protein Database (PDB) identification code 1ucu, is displayed in PROTEIN EXPLORER.(B) Table listing flagellin chimeras made and the effective concentration required for 50% maximal TLR5 activation in ng͞ml (EC50) Ϯ SD. A linear schematic of FliC shows the amino acid numbers of the do- mainboundaries.ND,notdetected.(C)Coo- massie-stained SDS͞PAGE of chimeric flagellins purified from Escherichia coli BL- 21RIL (1 ␮g of protein loaded per lane). The left margin shows molecular size in kDa. (D) Dose-response curve of CHO-hTLR5 to pu- rified flagellin chimeras as in 1A.

that we previously determined also plays a role in TLR5 recogni- the convex surface amino acids 89–96 (yellow, Fig. 4A). We tion. Amino acid 411 is an isoleucine in Salmonella typhimurium therefore mutated residues 58 and 59 of Salmonella typhimurium FliC, and, in our previous alanine-scanning mutagenesis study, FliC to the H. pylori FlaA sequence (K58S and G59S). The 58͞59 mutation of this residue in FliC to alanine caused the greatest mutations in FliC did not affect motility or TLR5 recognition reduction in TLR5 recognition and completely abolished bacterial (Figs. 4 B–D). However, mutations 58͞59 completely restored motility (3). All of the ␧ Proteobacteria flagellins have an alanine at motility to FliC I411A, while preserving reduced TLR5 activity position 411. Significantly, FliC isoleucine 411 is buried just below (Figs. 4 B–D). amino acids 89–96 in the convex surface (Fig. 4A, red). Our data This series of mutations suggests a mechanism by which a suggest that the FliC I411A mutation affects TLR5 recognition and TLR5-stimulatory flagellin could mutate to reduce its TLR5 ac- motility by changing the conformation of the exposed residues in tivity, while retaining motility. The first mutations would alter the the 89–96 region (Fig. 4A, yellow) (3). As expected, the FliC I411A contact surface but not change motility or TLR5 activity, as did the mutation reduced TLR5 activation and abrogated bacterial motility K58S͞G59S mutation, and would permit a subsequent mutation (Figs. 4 B–D). that would reduce TLR5 activity without affecting motility. Several Comparison of the concave surface revealed that Salmonella cycles of this process could result in the complex series of changes typhimurium and H. pylori flagellin differ in amino acid residues and the loss of TLR5 recognition that we observe in the ␣ and ␧ 58 and 59 (Fig. 4A, light blue), which in FliC interact directly with Proteobacteria flagellin molecules.

Fig. 3. Amino acids 89–96 are re- quired for TLR5 activation. (A) Se- quence alignment (CLUSTALW)of flagellin proteins from bacteria that activate TLR5 with those of the ␣ and ␧ Proteobacteria. Asterisks indicate residues in this region pre- viously determined to be important for TLR5 recognition (3). (B) Table listing flagellin chimeras made and their corresponding EC50 Ϯ SD. ND, not detected. (C) Dose-response curve of CHO-hTLR5 to purified flagellin chimeras. (D) Salmonella typhimurium BC696 (SL1344 fliC- fljB-) expressing wild-type or the 89–96 FlaA FliC flagellin chimera was stab-inoculated into motility agar and photographed. Data are representative of three indepen- dent experiments.

9250 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502040102 Andersen-Nissen et al. Downloaded by guest on October 3, 2021 Fig. 4. ␧ Proteobacteria possess compensatory changes that allow fila- ment formation and mo- tility. (A) Stacking of FliC monomers using PDB file 1io1 was performed ac- cording to Samatey et al. (24) and is represented in PROTEIN EXPLORER to show contact surfaces. (A Left) View of two monomers: dark blue, concave con- tact surface; green and yellow, convex contact surface; yellow, amino ac- ids 89–96. (A Right) Close-up of contact sur- face showing the location of 89–96 (yellow), I411 (red), and K58,G59 (light blue). Amino acids N87, L88, and R118 are re- presented as sticks to show the buried residue, I411. (B) Table listing mutant flagellins made and their corresponding EC50 Ϯ SD. (C) Dose-response curves of CHO-hTLR5 stimulated with purified mutant flagellins. (D) Salmonella typhimurium BC696 (SL1344 fliCϪfljBϪ) expressing wild-type or FliC mutant constructs were stab-inoculated into motility agar and photographed. Data are representative of three independent experiments.

Discussion carrier state, which facilitates fecal–oral spread of the bacteria and In this study, we demonstrate that members of the ␣ and ␧ the persistence of the bacteria within human populations. H. pylori Proteobacteria, including three important human pathogens, C. also has hypostimulatory LPS (32, 33), suggesting that structural jejuni, H. pylori, and Bartonella bacilliformis, possess flagellin modification of other pathogen-associated molecular patterns may molecules that cannot be recognized by TLR5. Their unique be important for its pathogenicity. ␣ flagellin sequences contain amino acid differences in the TLR5 The Proteobacterium Bartonella bacilliformis is transmitted by recognition site that permit TLR5 evasion, as well as compen- sandflies, but humans are its natural reservoir (34). It is a highly satory mutations that preserve bacterial motility. motile pathogen that invades and multiplies within red blood cells, This study independently confirms the location of the TLR5 causing the diseases Oroya and verruga peruana (34). Its recognition site on flagellin. Flagellin’s TLR5-stimulatory activity flagella are required for efficient binding and invasion of human red lies predominantly in the N-terminal D1 domain, centered around blood cells (35), suggesting that TLR5 evasion may also be relevant amino acids 89–96, but requires additional contribution from the to its pathogenesis. D2-D3 and the C-terminal D1 domain. Flagellin is a good adjuvant (e.g. see ref. 26), and this study and our previous report (3) clearly demonstrate that flagellin’s adjuvant activity is contained entirely within the amino acid sequence and is easily amenable to manip- ulation. Our studies indicate that proper folding and three- dimensional structure are critical for TLR5 recognition of flagellin, and must be taken into account when designing flagellin-based adjuvants for vaccines. Identification of sequences that disrupt proper stacking of flagellin monomers in the filament also points to potential sites for drug targets. IMMUNOLOGY Intriguingly, the bacteria that have flagellin amino acid changes that evade TLR5 recognition also require flagellar-based motility for infection or colonization. The ␧ Proteobacteria C. jejuni, H. pylori, and Helicobacter mustelae all require motility for colonization of their mammalian hosts (10–16). Once inside, they inhabit a variety of mucosal surfaces, including the stomach (H. pylori) and the small and large intestine (C. jejuni and H. hepaticus) where they are in close proximity to the epithelial cell layer (27–29). Another ␧ Proteobacterium, W. succinogenes, is a commensal that on mucosal surfaces of the bovine rumen (30). We predict that evasion of TLR5 recognition confers a selective advantage to the ␧ Pro- teobacteria, because epithelial cells recognize bacterial flagellin and Fig. 5. Some members of the ␣ Proteobacteria are predicted to be recog- activate proinflammatory responses that may prevent colonization nized by TLR5. Sequence alignment (CLUSTALW) of flagellin proteins from of mucosal surfaces (18, 19). In addition to causing disease, many bacteria that activate TLR5 with those of the ␣ and ␧ Proteobacteria in the individuals are asymptomatic carriers of H. pylori and C. jejuni (8, 89–96 region. Asterisks indicate residues in this region previously determined 31). TLR5 evasion may be critical for establishing this asymptomatic to be important for TLR5 recognition (3).

Andersen-Nissen et al. PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 ͉ 9251 Downloaded by guest on October 3, 2021 Some of the ␤ and ␥ clades seem to have stimulatory flagellin, it is likely that the TLR5-nonstimulatory developed strategies to down-regulate flagellin expression, rather flagellin arose within the ␣ clade and that the primordial flagellin than mutate their flagellin molecules to a form that is not detected most closely resembles the ␤͞␥ sequence. The conserved ␧ Pro- by TLR5. Some strains of Listeria monocytogenes down-regulate teobacteria flagellin 89–96 sequence is unique from the ␣ clade, flagellin at 37°C (36), whereas Salmonella typhimurium turns off suggesting that it arose independently during evolution. Flagellin flagellin production only after entering host phagocytes (37) after sequence data for the diverse array of ␧ Proteobacteria is currently breaching the epithelial barrier during invasion of the gastrointes- limited to pathogens and commensals that live at mucosal tinal tract. Escherichia coli and Pseudomonas species form biofilms sites, but this clade is actually comprised of a diverse array of in vivo and during this process down-regulate flagellin expression bacteria, including species that live in hydrothermal vents (44). As (38, 39). This mechanism averts TLR5 recognition, but also elim- more ␧ clade flagellin sequences become available, we predict that inates flagellar-based motility. The retention of flagellar-based other members will possess flagellin molecules that share the 89–96 motility in the ␣ and ␧ clades of Protobacteria presumably gives them sequence with the ␤ and ␥ clades, indicating that the TLR5- a selective advantage for their ecological niche and allows them to nonstimulatory ␧ Proteobacterial flagellin also arose within this compete efficiently in hosts with flagellin receptors. clade after the divergence of the Proteobacteria. The other member of the ␣ Proteobacteria that we found to evade An intriguing question that remains is which selective force drove TLR5, Rhizobium (Ensifer) meliloti, is a nitrogen-fixing sym- the independent selection of TLR5-nonstimulatory flagellin in biont that forms root nodules, but is not known to infect animals subsets of the ␣ and ␧ Proteobacteria. TLR5 is evolutionarily (40). Motility in this bacterium confers a competitive advantage for conserved within vertebrates, and has been demonstrated to rec- root colonization (40). This symbiont, as well as ognize flagellin in species as diverse as man (20) and rainbow trout tumefaciens, a plant pathogen whose flagella also increase virulence (45). Genetic studies indicate that the specificity of vertebrate (41), both evade recognition by FLS2, the plant defense receptor for TLR5 arose before the Cambrian period (ref. 46 and J. Roach, flagellin (42). FLS2 recognizes a linear site distinct from the TLR5 personal communication). Thus, TLR5 and its functional recogni- recognition site that is present in plant pathogens such as Pseudo- tion of flagellin has had sufficient time to influence the evolution ␥ monas syringae, a member of the Proteobacteria (42, 43). The of the ␣ and ␧ Proteobacterial flagellins. Alternatively, other forces FLS2 recognition site is a highly conserved 15-amino acid peptide in nature may have selected for these flagellin variants, which that spans portions of the N-terminal S and D1 domains (42, 43), serendipitously provided these bacteria with selective growth ad- and is located adjacent to the concave contact surface of flagellin vantages at mucosal sites. Regardless of the evolutionary history, (23). Like the TLR5 recognition site, this peptide also contacts identification of flagellin molecules present in human pathogenic adjacent monomers in the protofilament (24), and thus is also bacteria and in a commensal of the bovine rumen that are not predicted to be important for flagellar protofilament formation and recognized by TLR5 suggests that TLR5 recognition at mucosal Rhizobium meliloti A tumefaciens bacterial motility. Both and . surfaces is an important host defense mechanism against some flagellin sequences contain changes that we also predict preserve pathogens. bacterial motility. An examination of published flagellin sequences in the 89–96 ␣ We thank D. Underhill, T. Hawn, E. Gold, A. Diercks, N. Yudkovsky, C. region reveals that not all Proteobacteria are predicted to evade Rosenberger, K. Kennedy, A. Ozinsky, and the A.A. laboratory. We also TLR5. sphaeroides and , both of thank R. Bonneau, J. Roach, N. Salama, S. Swanzy, and C. Verlinde. This which live in aquatic environments, possess conserved amino acids work was supported by a predoctoral training grant from the Cancer of the ␤ and ␥ clades in the 89–96 region (Fig. 5). Because the ␣ Research Institute (to E.A.-N.), National Institutes of Health (NIH) Grant clade comprises bacteria that make TLR5-stimulatory and non- RO1 AIO52286 (to A.A.), and NIH Grant RO1 AI47242 (to B.T.C.).

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