Molecular Characterization of Two Toll Receptors in Hyriopsis cumingii and Their Potential Roles in Antibacterial Response.

Ying Huang,Qian Ren,Guosong Zhang

doi:10.3389/fphys.2019.00952

Ying Huang, Qian Ren + Show 1 more

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https://doi.org/10.3389/fphys.2019.00952

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Abstract

Tolls/Toll-like receptors (TLRs) play a key role in innate immunity by detecting the invading microbes and subsequently activating downstream signaling cascades. In this study, two new molluscan Toll members (designed as HcToll6 and HcToll7) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). The obtained HcToll6 full-length cDNA was 3207 bp consisting of a 2223 bp open reading frame (ORF) that encoded a peptide of 740 amino acids. HcToll7 cDNA is a 3216 bp molecule that contains an ORF of 2139 bp encoding a protein of 712 amino acids. The deduced HcToll6 and HcToll7 proteins share two common structures: extracellular leucine-rich repeat (LRR) domains and intracellular Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time PCR results showed that HcToll6 and HcToll7 were mainly expressed in the hepatopancreas and the gills, and they responded rapidly to bacterial stimulation. RNA interference by dsRNA results revealed that HcToll6 and HcToll7 RNAi strongly decreased the expression of lysozyme (HcLyso) and defensin (HcDef) in the gills of RNAi-treated mussels with Vibrio parahaemolyticus challenge. As a pattern recognition receptor, the prokaryotic expressed the recombinant LRR domains of HcToll6 and HcToll7 (rHcToll6-LRR and rHcToll7-LRR) could bind to Gram-positive and Gram-negative bacteria and had a strong tendency to recognize lipopolysaccharide (LPS) and peptidoglycan (PNG). rHcToll6-LRR and rHcToll7-LRR exhibited a significant in vitro bactericidal activity against V. parahaemolyticus and Staphylococcus aureus. These findings provide useful information to characterize Tolls in mussels.

Highlights

Lacking a true adaptive immune system, invertebrates against infectious agents mainly rely on innate immunity (Loker et al, 2004)
The full-length HcToll6 cDNA was 3207 bp, and it consisted of a 2223 bp open reading frame (ORF) that encoded a peptide of 740 amino acids, a 52 bp 5 untranslated region (UTR), and a 932 bp 3 UTR with a potential polyadenylation signal (AATAAA)
SMART analysis predicted that the deduced HcToll6 protein contained 12 leucine-rich repeat (LRR) residues, including nine 21–30 aa LRRs, a 32 aa LRR N-terminal domain (LRR-NT), a 24 aa LRR typical subfamily domain (LRR-TYP), and a 55 aa LRR C-terminal domain (LRR-CT) in the extracellular domain, a 23 aa transmembrane domain, and a 141 aa intracellular Toll/interleukin-1 receptor (TIR) domain (Figure 1)

Summary

Introduction

Lacking a true adaptive immune system, invertebrates against infectious agents mainly rely on innate immunity (Loker et al, 2004). Immune deficiency (IMD), and Janus family tyrosine kinase and signal transducer and activator of transcription (JAK/STAT) pathways are regarded as the three main pathways regulating the immune response of invertebrates (Li and Xiang, 2013). Spätzle binds to Toll receptor and mediates downstream molecules containing Myeloid differentiation factor 88 (MyD88), Tube, Pelle, tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), Cactus, and Dorsal (Anderson, 2000; Silverman and Maniatis, 2001). These cascades activate NF-κB transcription factors, which regulate the synthesis of specific target genes, such as antibacterial peptide genes (AMPs) (Lemaitre and Hoffmann, 2007)

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