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A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin.

Andersen-Nissen E, Smith KD, Bonneau R, Strong RK, Aderem A - J. Exp. Med. (2007)

Bottom Line: Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition.These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand.These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

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Mouse TLR5 detects most flagellins better than human TLR5. (A) Immunoblot of CHO cells stably expressing vector alone or human (h) or mouse (m) TLR5. 100 μg of cellular cytoplasmic extracts were loaded per lane, and TLR5 expression was detected by immunoblotting for the HA epitope tag. Equal loading was verified by immunoblotting for β-tubulin. Kilodalton values are shown. (B–F) Shown is the percent fold induction of NF-κB luciferase activity for flagellin purified from each indicated bacterial species relative to maximal stimulation achieved with S. typhimurium flagellin for cells expressing either human (continuous line) or mouse (dashed line) TLR5 at the indicated flagellin doses. Data are representative of at least three independent experiments. Error bars represent the mean ± SD. (G) Table listing the effective flagellin concentrations needed to achieve half maximal activation of TLR5 (EC50). p-values were calculated using a two-tailed Student's t test.
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fig1: Mouse TLR5 detects most flagellins better than human TLR5. (A) Immunoblot of CHO cells stably expressing vector alone or human (h) or mouse (m) TLR5. 100 μg of cellular cytoplasmic extracts were loaded per lane, and TLR5 expression was detected by immunoblotting for the HA epitope tag. Equal loading was verified by immunoblotting for β-tubulin. Kilodalton values are shown. (B–F) Shown is the percent fold induction of NF-κB luciferase activity for flagellin purified from each indicated bacterial species relative to maximal stimulation achieved with S. typhimurium flagellin for cells expressing either human (continuous line) or mouse (dashed line) TLR5 at the indicated flagellin doses. Data are representative of at least three independent experiments. Error bars represent the mean ± SD. (G) Table listing the effective flagellin concentrations needed to achieve half maximal activation of TLR5 (EC50). p-values were calculated using a two-tailed Student's t test.

Mentions: We expressed epitope-tagged human or mouse TLR5 and NF-κB luciferase reporter constructs in CHO-K1 cells, and similar TLR5 expression levels were detected in the two stably transfected populations (Fig. 1 A). We purified flagellin from Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Serratia marcescens and tested the ability of each flagellin to stimulate either human or mouse TLR5. Mouse TLR5 detected S. typhimurium, E. coli, P. aeruginosa, and L. monocytogenes flagellins at lower doses than human TLR5 (Fig. 1, B–E). These differences could not be explained by variability in receptor expression or signaling, as human TLR5 recognized S. marcescens flagellin better than mouse TLR5 (Fig. 1 F). The effective concentrations of flagellin that elicit 50% maximal response (EC50) for human and mouse TLR5 are listed in Fig. 1 G.


A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin.

Andersen-Nissen E, Smith KD, Bonneau R, Strong RK, Aderem A - J. Exp. Med. (2007)

Mouse TLR5 detects most flagellins better than human TLR5. (A) Immunoblot of CHO cells stably expressing vector alone or human (h) or mouse (m) TLR5. 100 μg of cellular cytoplasmic extracts were loaded per lane, and TLR5 expression was detected by immunoblotting for the HA epitope tag. Equal loading was verified by immunoblotting for β-tubulin. Kilodalton values are shown. (B–F) Shown is the percent fold induction of NF-κB luciferase activity for flagellin purified from each indicated bacterial species relative to maximal stimulation achieved with S. typhimurium flagellin for cells expressing either human (continuous line) or mouse (dashed line) TLR5 at the indicated flagellin doses. Data are representative of at least three independent experiments. Error bars represent the mean ± SD. (G) Table listing the effective flagellin concentrations needed to achieve half maximal activation of TLR5 (EC50). p-values were calculated using a two-tailed Student's t test.
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fig1: Mouse TLR5 detects most flagellins better than human TLR5. (A) Immunoblot of CHO cells stably expressing vector alone or human (h) or mouse (m) TLR5. 100 μg of cellular cytoplasmic extracts were loaded per lane, and TLR5 expression was detected by immunoblotting for the HA epitope tag. Equal loading was verified by immunoblotting for β-tubulin. Kilodalton values are shown. (B–F) Shown is the percent fold induction of NF-κB luciferase activity for flagellin purified from each indicated bacterial species relative to maximal stimulation achieved with S. typhimurium flagellin for cells expressing either human (continuous line) or mouse (dashed line) TLR5 at the indicated flagellin doses. Data are representative of at least three independent experiments. Error bars represent the mean ± SD. (G) Table listing the effective flagellin concentrations needed to achieve half maximal activation of TLR5 (EC50). p-values were calculated using a two-tailed Student's t test.
Mentions: We expressed epitope-tagged human or mouse TLR5 and NF-κB luciferase reporter constructs in CHO-K1 cells, and similar TLR5 expression levels were detected in the two stably transfected populations (Fig. 1 A). We purified flagellin from Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Serratia marcescens and tested the ability of each flagellin to stimulate either human or mouse TLR5. Mouse TLR5 detected S. typhimurium, E. coli, P. aeruginosa, and L. monocytogenes flagellins at lower doses than human TLR5 (Fig. 1, B–E). These differences could not be explained by variability in receptor expression or signaling, as human TLR5 recognized S. marcescens flagellin better than mouse TLR5 (Fig. 1 F). The effective concentrations of flagellin that elicit 50% maximal response (EC50) for human and mouse TLR5 are listed in Fig. 1 G.

Bottom Line: Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition.These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand.These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

Show MeSH
Related in: MedlinePlus