Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro.
The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.
Murthy
Kanneganti G K
KG
Inotek Pharmaceuticals Corp., Beverly, Massachusetts 01915, USA.
Deb
Amitabha
A
Goonesekera
Sunali
S
Szabó
Csaba
C
Salzman
Andrew L
AL
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005408:Flagellin / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation; D016209:Interleukin-8 / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012475:Salmonella / Q000378:metabolism*; D013997:Time Factors; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D014162:Transfection
D018076:DNA, Complementary; D016209:Interleukin-8; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D005408:Flagellin; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide