Fig. 1.
Flagellin from α and ε Proteobacteria is not detected by TLR5. (A and B) Approximately 1 × 105 CHO cells stably expressing human TLR5 and an NF-κB 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 (≈10- 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-κB 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 ≈23 and 26 kDa likely represent degraded flagellin). (Right) ε Proteobacteria flagellins as detected with anti-C. jejuni flagellin antiserum (additional band in H. pylori lanes likely represents crossreacting hook protein ≈75 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 SDS/PAGE 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. -, flagellated bacteria tested that did not activate TLR5; *, flagellated bacteria tested that activated TLR5.