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
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
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DOI：
10.1074/jbc.m307759200
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发表时间：
2004-02-13
影响因子：
4.8
通讯作者：
Salzman, AL
中科院分区：
文献类型：
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作者：
Murthy, KGK;Deb, A;Salzman, AL
细菌表面蛋白鞭毛蛋白在远缘细菌物种中广泛分布并且很好地保守。我们和其他研究人员最近报道，纯化的鞭毛蛋白从都柏林沙门氏菌或重组鞭毛蛋白的慕尼黑沙门氏菌的起源结合到真核细胞的Toll受体TLR 5和激活核转位的NF-κ B和丝裂原活化蛋白激酶，导致在体外和体内释放的促炎介质的主机。不同革兰氏阴性菌鞭毛蛋白的氨基酸序列比对表明，C和N-末端是很保守的。N和C末端或两者内的序列可能调节鞭毛蛋白的促炎活性。在这里，我们开始更精确地绘制TLR 5激活和促炎信号所需的两端区域。从任一末端系统性缺失氨基酸逐渐减少真核促炎激活。然而，删除氨基酸95 - 108（基序N）在N端和441-449（基序C）在C端完全废除促炎活性。定点突变分析进一步证明了基序N和C的重要性。我们还提出了证据的功能作用的基序N和C与TLR 5受体使用报告分析系统。总之，我们的结果表明，鞭毛蛋白的促炎活性的结果从基序N与TLR 5受体的细胞表面上的相互作用。
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.