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)-Lys4 trihydrochloride (Pam3CSK4) 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 (Pam3CSK4) 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 Pam3CSK4 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 H2O2-MAPK-p38–dependent axis is crucial for regulation of TLR2 gene expression in fish macrophages.

Cloning and expression of Toll-like receptors 1 and 2 from a teleost fish, the orange-spotted grouper Epinephelus coioides

“…Although the bacterial number was not quantified in the V. alginolyticus-infected fish, the increased expression of TLR1 and TLR2, as well as MyD88 genes in spleen and kidney of the orange-spotted groupers injected with V. alginolyticus and with LPS and Poly(I:C) may in fact imply, as shown in Japanese flounder and in zebrafish (Takano et al, 2006;Liu et al, 2010), the activation of MyD88-dependent pathway in the grouper, leading to the induction of IL-1␤. Recent study also reported the increased expression of IL-1␤ via TLR2 in carp macrophages when stimulated with peptidoglycan (Ribeiro et al, 2010).…”

“…In teleost fish, TLR6 and TLR10 are not identified, and TLR4 is only found in pufferfish and zebrafish as well as in lamprey (Lampetra japonica) (Ishii et al, 2007). But unlike in mammal, TLR4 in zebrafish does not recognize LPS (Sepulcre et al, 2009); and recent research has shown that TLR2 in fish may be incorporated with PGRP (Chang and Nie, 2008), and can indeed bind lipoteichoic acid and peptidoglycan, as mammalian TLR2 (Ribeiro et al, 2010). Ribeiro et al (2010) also showed that fish TLR2 expression can be regulated via MAPK-p38 activation.…”

Carp Il10 Has Anti-Inflammatory Activities on Phagocytes, Promotes Proliferation of Memory T Cells, and Regulates B Cell Differentiation and Antibody Secretion

“…MACS was used in combination with carp leukocyte-specific Abs to isolate neutrophils from mid-kidney leukocytes and B cells from PBLs, as previously described (30,(34)(35)(36). The purity of the sorted leukocytes was .98%, as confirmed by flow cytometry using a BD FACSCanto A (BD Biosciences).…”

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10

“…MACS was used in combination with carp leukocyte-specific Abs to isolate carp neutrophils from mid-kidney leukocytes, and carp B cells from PBL as previously described (13,(17)(18)(19). The purity of the sorted leukocytes was .98% as confirmed by flow cytometry using a BD FACSCanto A (BD Biosciences).…”