Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii.

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

Characterization of a novel toll-like receptor and activation NF-κB signal pathway in triangle sail mussel Hyriopsis cumingii.

Toll-like receptor (TLR) family plays an important role in innate immunity for detection of and defense against microbial pathogens. In this study, a novel toll-like receptor (HcTLRn) was characterized from freshwater pearl mussel H. cumingii. The complete sequence of HcTLRn was 3725 bp, and the open reading frame (ORF) encoded 718 amino acid residues. Predicted HcTLRn protein possessed seven atypical leucine-rich repeat (LRR) domains, two typical LRR subfamily domains, a C-terminal domain LRR, a transmembrane domain and an intracellular Toll/interleukin-1 (IL-1) receptor domain. Transcripts of HcTLRn were constitutive expressed in the tissues of healthy mussels and were markedly induced in hepatopancreas and gills after lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic polycytidylic acid (ploy I: C) stimulation. Knockdown of HcTLRn in vivo significantly decreased the mRNA levels of TLR pathway transcription factors p65 and p105 as well as antimicrobial peptides (AMPs) including lysozyme (HcLys), theromacin (HcTher), whey acidic protein (HcWAP), LPS-binding protein/bactericidal permeability increasing protein (HcLBP/BPI) 1 and 2 after mussels challenged by LPS. In situ hybridization results showed that HcTLRn mRNA was significantly increased in hemocytes after LPS, PGN and poly I:C stimulation. HcTLRn protein was mainly expressed in hepatopancreas and gills and was significantly increased after LPS stimulation. Moreover, recombinant extracellular domain of HcTLRn (HcTLRn-ECD) proteins could bind to a variety of bacterial and pathogen-associated molecular patterns such as LPS, PGN, and poly I:C in vitro. Subcellular localization results showed that HcTLRn was mainly distributed near the cell membrane and in cytoplasm. Over-expression of HcTLRn activated the NF-κB luciferase reporter in HEK293T cells. Collectively, these results suggested that HcTLRn was a TLR family member that might play an important role in activation of NF-κB signal pathway in Mollusca.