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J. Biol. Chem., Vol. 279, Issue 7, 5667-5675, February 13, 2004

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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^*

Kanneganti G. K. Murthy, Amitabha Deb, Sunali Goonesekera, Csaba Szabó, and Andrew L. Salzman

From the Inotek Pharmaceuticals Corp., Beverly, Massachusetts 01915

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-B 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.

Received for publication, July 17, 2003 , and in revised form, September 5, 2003.

^* This work was supported by National Institutes of Health Grants R01GM60699 and R43AI48249. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inotek Pharmaceuticals Corp., 100 Cummings Center, Ste. 419E, Beverly, MA 01915. Tel.: 978-232-9660; Fax: 978-232-8975; E-mail: alsalzman{at}aol.com.

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