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*Department of Biochemistry, Microbiology, and Molecular Biology,
Department of Chemical and Biological Engineering and the Laboratory for Surface Science and Technology, and
Graduate School of Biomedical Sciences, University of Maine, Orono, ME 04469;
Department of Computer and Information Science and
¶Institute of Neuroscience, University of Oregon, Eugene, OR 97403
Mammalian immune responses to LPS exposure are typified by the robust induction of NF-
B and IFN-β responses largely mediated by TLR4 signal transduction pathways. In contrast to mammals, Tlr4 signal transduction pathways in nontetrapods are not well understood. Comprehensive syntenic and phylogenetic analyses support our hypothesis that zebrafish tlr4a and tlr4b genes are paralogous rather than orthologous to human TLR4. Furthermore, we provide evidence to support our assertion that the in vivo responsiveness of zebrafish to LPS exposure is not mediated by Tlr4a and Tlr4b paralogs because they fail to respond to LPS stimulation in vitro. Zebrafish Tlr4a and Tlr4b paralogs were also unresponsive to heat-killed Escherichia coli and Legionella pneumophila. Using chimeric molecules in which portions of the zebrafish Tlr4 proteins were fused to portions of the mouse TLR4 protein, we show that the lack of responsiveness to LPS was most likely due to the inability of the extracellular portions of zebrafish Tlr4a and Tlr4b to recognize the molecule, rather than to changes in their capacities to transduce signals through their Toll/IL-1 receptor (TIR) domains. Taken together, these findings strongly support the notion that zebrafish tlr4a and tlr4b paralogs have evolved to provide alternative ligand specificities to the Tlr immune defense system in this species. These data demonstrate that intensive examination of gene histories when describing the Tlr proteins of basally diverging vertebrates is required to obtain fuller appreciation of the evolution of their function. These studies provide the first evidence for the functional evolution of a novel Tlr.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Grant R15AI049237-02 to C.S., C.R.L., and C.H.K. from the National Institute for Allergy and Infectious Disease (NIAID), Grant R01 RR10715 to J.H.P. from the National Center for Research Resources (NCRR), and Grant R15AI065509-01 to P.J.M. from NIAID. All institutes are components of the National Institutes of Health (NIH), and the contents of the article are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.
2 Current address: The Jackson Laboratory, Bar Harbor, ME 04609.
3 Current address: Department of Biology, University of Maine at Augusta, Augusta, ME 04330.
4 Address correspondence and reprint requests to Dr. Carol H. Kim, Department of Biochemistry, Microbiology, and Molecular Biology, 5735 Hitchner Hall, University of Maine, Orono, ME 04469. E-mail address: carolkim{at}maine.edu
5 Abbreviations used in this paper: LBP, LPS-binding protein; TIR, Toll/IL-1 receptor; LRR, leucine-rich repeat; SHRV, snakehead rhabdovirus; dpf, days postfertilization; hpe, hours postexposure; ZFL, zebrafish liver cells; mx, myxovirus (influenza virus) resistance gene; qRT-PCR, quantitative RT-PCT; HKEC, heat-killed Escherichia coli; HKLP, heat-killed Legionella pneumophila; TICAM, TIR domain-containing adaptor molecule.
6 The online version of this article contains supplemental material.
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