Cyprinid‐specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors

Abstract

Membrane‐embedded Toll‐like receptor 5 (TLR5) functions as a homodimer to detect bacterial flagellin. Cyprinid grass carp (Ctenopharyngodon idella) encodes two TLR5 genes, CiTLR5a and CiTLR5b. Here, we show that cyprinid TLR5a and TLR5b homodimers unexpectedly bind the dsRNA analog poly(I:C) and regulate interferon (IFN) response in early endosomes and lysosomes. Although TLR5 homodimers also bind flagellin, an immune response to flagellin is only triggered by TLR5a/b heterodimer. Moreover, we demonstrate that two TLR5 paralogs have opposite effects on antiviral response: CiTLR5a slightly promotes and powerfully maintains, whereas CiTLR5b remarkably inhibits virus replication. We show that the ectodomain of CiTLR5 is required for dsRNA‐induced IFN signaling, and we map the key poly(I:C) binding sites to G240 for CiTLR5a and to N547 for CiTLR5b. Furthermore, we reveal that differential N‐glycosylation of CiTLR5a/b affects dsRNA‐IFN signaling but has no role in flagellin‐mediated NF‐κB induction, with paralog‐specific roles for CiTLR5a‐T101 and corresponding CiTLR5b‐I99. Moreover, we provide evidence that the ability to sense dsRNA represents a neofunctionalization specific for membrane‐bound TLR5 in cyprinid, bridging viral and bacterial immune responses.

Genome-wide identification and characterization of toll-like receptor 5 (TLR5) in fishes

“…After A. hydrophila infection, overexpression of CiTLR5Ma and CiTLR5Mb genes were observed in immune system tissues and vital organs, including the liver, spleen, and head kidney, suggestive of their important roles in the immune response to the invasion of pathogens. Moreover, CiTLR5Ma and CiTLR5Mb showed similar expression patterns across tissues, which supports the finding that CiTLR5Ma and CiTLR5Mb are subfunctionalized to sense bacterial flagellin as a heterodimer (Liao et al, 2022).…”

“…Frontiers in Genetics frontiersin.org soluble members of the M/S type play a synergistic role in sensing flagellin (Tsujita et al, 2004) possibly implying that soluble TLR5 is redundant in function and disappears in some fish and endothermic vertebrates. Cyprinid-specific duplicated membrane TLR5 (M/M type) demonstrated neofunctionalization to sense viral dsRNA as functional homodimeric receptors in antagonistic effect (Liao et al, 2022). Domain number variation was identified in the fish TLR5 isotypes, generating functional diversity and complexity (Asami and FIGURE 4 The expression patterns of CiTLR5Ma and CiTLR5Mb on transcription level in tissues of liver, spleen and head kidney, after infected with A.…”

“…TLRs are antigenrecognition receptors on the surfaces or inside cells that directly recognize pathogenassociated molecular patterns (PAMPs) (Mogensen, 2009). All TLRs have a leucine-rich repeat (LRR) ectodomain for PAMP recognition, transmembrane (TM) domain for TLR dimerization and stabilization, and toll/IL-1 receptor (TIR) domain associated with signaling (Brubaker et al, 2015;Voogdt et al, 2016;Voogdt et al, 2018;Su and Gao et al, 2022;Liao et al, 2022). According to the previous data, at least 13 TLR types have been reported in mammals (Janeway and Medzhitov, 2002;Akira et al, 2006); for example, 12 (TLR1-9, TLR11-13) and 10 (TLR1-10) TLR genes have been reported in the mice and human genome, respectively.…”

Fish TLR5 develops a taste for viral RNA

“…Teleost fish have undergone whole genome duplication and grass carp encodes two tandem TLR5 genes, CiTLR5a and CiTLR5b . Liao et al ( 2022 ) started by comparing the sensitivity of the two CiTLR5 paralogs to different MAMPs. Various flagellins or flagellated bacteria were sensed by CiTLR5a and CiTLR5b heterodimers for activation of NF‐κB, a transcription factor regulating the expression of pro‐inflammatory genes in both fish and mammals.…”

“…In mammals, TLR3 is specialized in sensing viral dsRNA (Liu et al, 2008), while TLR5 recognizes the MAMP flagellin (Yoon et al, 2012;Fig 1). The well-established notion of TLR5 as a purely "bacterial" flagellin TLR has now been challenged by Liao et al in this issue of EMBO Reports (Liao et al, 2022) Notably, the CiTLR5 ectodomains directly bound poly(I:C). Through a comprehensive series of domain swaps and mutagenesis, the authors showed that flagellin recognition requires canonical TLR5 recognition sites in both CiTLR5a and CiTLR5b.…”