Evolution of Recognition of Ligands from Gram-Positive Bacteria: Similarities and Differences in the TLR2-Mediated Response between Mammalian Vertebrates and Teleost Fish

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands.
The Journal of Immunology (Impact Factor: 4.92). 03/2010; 184(5):2355-68. DOI: 10.4049/jimmunol.0900990
Source: PubMed


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 3CSK 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 3CSK 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 3CSK 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 2O 2-MAPK-p38-dependent axis is crucial for regulation of TLR2 gene expression in fish macrophages.

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Available from: Geert F Wiegertjes
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    • "Of interest, out of eight accessory molecules classified in mammals as important mediators of ligand delivery and/or recognition and required for TLR function (Lee et al., 2012) only four molecules, including CD36, seem to have clear orthologs in fish genomes (Pietretti et al., 2013). Given our interest in the function of TLRs (Pietretti and Wiegertjes, 2014) among which Tlr2 (Ribeiro et al., 2010), we studied Cd36 in zebrafish and common carp, two closely related cyprinid fish species, taking advantage of the complementary tools available for these two species to achieve a more thorough understanding of the function of Cd36 in teleost fish. We characterize for the first time in detail Cd36 of zebrafish and two Cd36 molecules of common carp finding a molecular structure consistent with mammalian CD36. "
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    ABSTRACT: CD36 is a scavenger receptor which has been studied closely in mammals where it is expressed by many different cell types and plays a role in highly diverse processes, both homeostatic and pathologic. It is among other things important in the innate immune system, in angiogenesis, and in clearance of apoptotic cells, and it is also involved in lipid metabolism and atherosclerosis. Recently, in the cephalochordate amphioxus a primitive CD36 family member was described, which was present before the divergence of CD36 from other scavenger receptor B family members, SCARB1 and SCARB2. Not much is known on the Cd36 molecule in teleost fish. We therefore studied Cd36 in both zebrafish and common carp, two closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp has two cd36 genes, and all show conserved synteny compared to mammalian CD36. The gene expression of carp cd36 is high in brain, ovary and testis but absent in immune organs. Although in mammals CD36 expression in erythrocytes, monocytes and macrophages is high, gene expression studies in leukocyte subtypes of adult carp and zebrafish larvae, including thrombocytes and macrophages provided no indication for any substantial expression of cd36 in immune cell types. Surprisingly, analysis of the cd36 promoter region does show the presence of several binding sites for transcription factors known to regulate immune responses. Overexpression of carp cd36 locates the receptor on the cell surface of mammalian cell lines consistent with the predicted topology of cyprinid Cd36 with a large extracellular domain, two transmembrane domains, and short cytoplasmic tails at both ends. Gene expression of cd36 is down-regulated during infection of zebrafish with Mycobacterium marinum, whereas knockdown of cd36 in zebrafish larvae led to higher bacterial burden upon such infection. We discuss the putative role for Cd36 in immune responses of fish in the context of other members of the scavenger receptor class B family.
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    • "TLR2 gene was found to be mainly expressed in peripheral blood leukocytes, in head kidney, spleen and gill [4] [17] [52]. The upregulation of teleost TLR2 expression following stimulation with lipopeptides from Gram-positive bacteria, have been demonstrated in P. olivaceus [17], D. rerio [34], E. coioides [52], and C. carpio [43]. In I. punctatus a Gram-negative bacterium, Edwardsiella ictaluri, seems to down-regulate TLR2 expression [4]. "
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    ABSTRACT: In the present study we address the investigation of TLR2 evolutionary selection in two Antarctic teleosts, Trematomus bernacchii (Nototheniidae) and Chionodraco hamatus (Channichthyidae). The nucleotide sequence of TLR2 has been determined in both species, encoding 20 leucine-rich repeats (LRRs) in the extracellular region and a classical Toll/IL-1R (TIR) domain in the intracellular region. High expression level of T. bernacchii TLR2 was found in spleen and skin. Using different methods we identified six codons that underwent Darwinian selection while 20 were found to be negatively selected. Molecular models of C. hamatus and T. bernacchii TLR2 ectodomain as well as of the TIR domain were built by Homology Modeling. Molecular Dynamics simulations were performed in water for 15 ns. The sites under positive selection were residing on the convex side of the solenoid, four out of six were in a 35-residue-long region including the central/N-terminal domain boundary: two in the external loop of LRR11 and the other two in the LRR12 loop. This region has been demonstrated to be the functional site of ligand interaction in human TLR2 structure. Antarctic TLR2 models showed more flexibility than TLR2 from the temperate species Gasterosteus aculeatus. These results suggest that the selective pressure has shaped TLR2 molecule in such a way that increased its activity under the peculiar Antarctic environmental conditions.
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    • "Only some of the zebrafish TLR ligands are currently known [29]. The specificity of TLR2, TLR3, and TLR5 is conserved between mammals and fish, recognizing lipopeptides, dsRNA, and flagellin, respectively [13, 30, 31]. Additionally, the fish-specific TLR22 has been shown to recognize dsRNA and PolyI:C [31]. "
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    ABSTRACT: The zebrafish has proven itself as an excellent model to study vertebrate innate immunity. It presents us with possibilities for in vivo imaging of host-pathogen interactions which are unparalleled in mammalian model systems. In addition, its suitability for genetic approaches is providing new insights on the mechanisms underlying the innate immune response. Here, we review the pattern recognition receptors that identify invading microbes, as well as the innate immune effector mechanisms that they activate in zebrafish embryos. We compare the current knowledge about these processes in mammalian models and zebrafish and discuss recent studies using zebrafish infection models that have advanced our general understanding of the innate immune system. Furthermore, we use transcriptome analysis of zebrafish infected with E. tarda, S. typhimurium, and M. marinum to visualize the gene expression profiles resulting from these infections. Our data illustrate that the two acute disease-causing pathogens, E. tarda and S. typhimurium, elicit a highly similar proinflammatory gene induction profile, while the chronic disease-causing pathogen, M. marinum, induces a weaker and delayed innate immune response.
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