Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mikiss).
Rainbow trout (Onchorhynchus mikiss) possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). Here we elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. The LRR regions of rtTLR5S and rtTLR5M exhibited 81% homology and relatively high (35.6 and 33.7%) homology to the extracellular domains of human TLR5 (huTLR5). Thus, two distinct genes encode the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR). In order to test their functions, we constructed fusion proteins with the LRR region of rtTLR5S (S-chimera) or that of rtTLR5M and the TIR of huTLR5 (M-chimera). The S- and M-chimeras expressed in HeLa or CHO cells signaled the presence of Vibrio anguillarum flagellin, resulting in NF-kappaB activation. rtTLR5M was ubiquitously expressed, whereas rtTLR5S was predominantly expressed in the liver. In the hepatoma cell lines of the rainbow trout RTH-149, stimulation of rtTLR5M with V. anguillarum or its flagellin allowed the up-regulation of rtTLR5S. Flagellin-mediated NF-kappaB activation was more significant in the presence of or simultaneous expression of rtTLR5S. Therefore, a two-step flagellin response occurred for host defense against bacterial infection in fish: (a) flagellin first induced basal activation of NF-kappaB via membrane TLR5, facilitating the production of soluble TLR5 and minimal acute phase proteins, and (b) the inducible soluble TLR5 amplifies membrane TLR5-mediated cellular responses in a positive feedback fashion.
Tsujita
Tadayuki
T
Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan.
Tsukada
Hironobu
H
Nakao
Miki
M
Oshiumi
Hiroyuki
H
Matsumoto
Misako
M
Seya
Tsukasa
T
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015139:Blotting, Southern; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D020224:Expressed Sequence Tags; D005408:Flagellin / Q000737:chemistry; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D017686:Oncorhynchus mykiss; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D014162:Transfection; D015854:Up-Regulation; D014733:Vibrio
D017931:DNA Primers; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D020033:Protein Isoforms; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D005408:Flagellin; D005982:Glutathione Transferase; D007930:Leucine