title: Evolution of recognition of ligands from Gram-positive bacteria: similarities and differences in the TLR2-mediated response between mammalian vertebrates and teleost fish.
abstract: We investigated the role of the TLR2 receptor in the recognition of ligands from Gram-positive bacteria in fish. Comparative sequence analysis showed a highly conserved Toll/IL-1 receptor domain. Although the leucine-rich repeat domain was less conserved, the position of the critical peptidoglycan (PGN)-binding residues in the leucine-rich repeat domain of carp TLR2 were conserved. Transfection of human embryonic kidney 293 cells with TLR2 corroborated the ability of carp TLR2 to bind the prototypical mammalian vertebrate TLR2 ligands lipoteichoic acid (LTA) and PGN from Staphylococcus aureus. The synthethic triacylated lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys(4) trihydrochloride (Pam(3)CSK(4)) but not the diacylated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) also activated TLR2 transfected human cells. We identified clear differences between the mammalian vertebrates and carp TLR2-mediated response. The use of the same ligands on carp macrophages indicated that fish cells require high concentrations of ligands from Gram-positive bacteria (LTA, PGN) for activation and signal transduction, react less strongly (Pam(3)CSK(4)) or do not react at all (MALP-2). Overexpression of TLR2 in carp macrophages confirmed TLR2 reactivity of the response to LTA and PGN, low-responsiveness to Pam(3)CSK(4) and nonresponsiveness to MALP-2. A putative relation with the apparent absence of accessory proteins such as CD14 from the fish TLR2-containing receptor complex is discussed. Moreover, activation of carp macrophages by PGN resulted in increased TLR2 gene expression and enhanced TLR2 mRNA stability, MAPK-p38 phosphorylation and increased radical production. Finally, we could show that NADPH oxidase-derived radicals and MAPK-p38 activation cooperatively determine the level of PGN-induced TLR2 gene expression. We propose that the H(2)O(2)-MAPK-p38-dependent axis is crucial for regulation of TLR2 gene expression in fish macrophages.
authors.affiliation: Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands.
authors.forename: Carla M S
authors.initials: CM
authors.lastname: Ribeiro
authors.forename: Trudi
authors.initials: T
authors.lastname: Hermsen
authors.forename: Anja J
authors.initials: AJ
authors.lastname: Taverne-Thiele
authors.forename: Huub F J
authors.initials: HF
authors.lastname: Savelkoul
authors.forename: Geert F
authors.initials: GF
authors.lastname: Wiegertjes
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002347:Carps / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008322:Mammals / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010457:Peptidoglycan / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000378:metabolism; D013682:Teichoic Acids / Q000378:metabolism / Q000494:pharmacology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014714:Vertebrates / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D029941:Fish Proteins; D008070:Lipopolysaccharides; D010457:Peptidoglycan; D013682:Teichoic Acids; D051195:Toll-Like Receptor 2; D049452:Green Fluorescent Proteins; C009900:lipoteichoic acid; D006861:Hydrogen Peroxide; D048051:p38 Mitogen-Activated Protein Kinases