Molecular Cloning and Functional Characterization of Chicken Toll-like Receptors

Abstract

Toll-like receptors (TLR) in the innate immune system have not been identified in non-mammalian vertebrates. Two types of TLR were cloned from a chicken bursa cDNA library using degenerate primers based on the consensus sequences of mouse and Drosophila Toll and designated as chicken TLR (chTLR) type 1 and type 2. Of the nine human TLRs reported to date, these chTLRs showed the highest homology to human TLR2. The extracellular regions of type 1 and type 2 contained a distinct ϳ200-amino acid stretch and were 45.3 and 46.3% homologous to that of human TLR2. The intracellular Toll/interleukin-1R homology domain of type 1 and type 2 was perfectly identical to each other and highly homologous (80.7%) to that of human TLR2. Both types were widely detected by reverse transcriptase-polymerase chain reaction and immunoblotting in various chicken organs, especially those rich in connective tissue. Both genes were mapped to chromosome 4q1.1, suggesting that they arose by gene duplication. By reporter gene assay, type 2 and to a lesser extent type 1, selectively signaled the presence of mycoplasma macrophage-activating lipopeptide-2/M161Ag in the human embryonic kidney 293 cell system. Cotransfection of type 2 and human CD14 or MD-2 into human embryonic kidney 293 cells allowed the response to Escherichia coli lipopolysaccharide (LPS), whereas type 1 did not signal LPS or any other microbial components tested. These results indicated that chTLR type 2 covers two major microbe patterns, lipoproteins and LPS, which are regulated by TLR2 and TLR4 in mammals. In oviparous animals, the duplicated TLRs in the pattern-recognition system may function for host-pathogen discrimination in a manner that is distinct from that in mammals.

Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily

“…5). We first confirmed that the chTLRs function in the human system using human HEK293 cells as reported previously (4). A single TLR member of the chTLR2 subfamily alone had no ability to induce luciferase activity in response to PGN, Malp-2 or Pam3.…”

“…This subfamily of TLRs appears to have advanced as a pattern recognition receptor in conformity with principles of evolution. We had identified chTLR2 type1 and chTLR2 type2 earlier than the chicken genome information was published (4).…”

“…Since chicken TLR2 consisted of duplicated isoforms with high similarity, we previously assigned their functional profiles according to contemporary knowledge (4). They responded to mycoplasmal diacylated lipoprotein, namely the macrophage-activating lipopeptide 2 kDa (Malp-2) leading to NF-κB activation (5,6), though the response was very weak (4).…”

“…They responded to mycoplasmal diacylated lipoprotein, namely the macrophage-activating lipopeptide 2 kDa (Malp-2) leading to NF-κB activation (5,6), though the response was very weak (4). Recently, Yilmaz et al, (7) have found from bioinformatics analysis of the chicken genome that chicken has a TLR2 subfamily consisting of chTLR1 type1 (1-1), chTLR1 type2 (1-2), chTLR2 type1 (2-1) and chTLR2 type2 (2-2).…”

Chicken Toll-like Receptor 3 Recognizes Its Cognate Ligand When Ectopically Expressed in Human Cells

“…However, prior to our work, it was demonstrated that ChTLR2 is functional when expressed in the human HEK293 cell line. 3 Our own experiments unambigu ously showed that ChTLR3 is active in HEK293 cells and that it exclusively recognizes dsRNA, like its mammalian counter part.…”