A triple WAP domain containing protein acts in antibacterial immunity of weather loach, Misgurnus anguillicaudatus.

Whey acidic protein domain (WAPD) occurs in a variety of proteins in animals and many of WAPD-containing proteins are involved in immunity. In the present study, a novel protein containing three WAPDs was identified from the weather loach, Misgurnus anguillicaudatus, designated as MaTWD. MaTWD share high identity with TWDs from fish but low identity with TWDs from other animal phyla. MaTWD transcripts mainly distributed in gills and head kidney responded to bacterial challenge with significant upregulation. In vitro assay with recombinant MaTWD protein revealed that MaTWD had antiprotease activity against bacterial proteases. Moreover, MaTWD exhibited bacterial binding capacity and antimicrobial activity. Most importantly, exogenous MaTWD protected loach against bacterial infection by reducing loach mortality. We infer that MaTWD participates in the antibacterial immunity of loach via its antiprotease and antimicrobial activities.

A C-type lectin with antibacterial activity in weather loach, Misgurnus anguillicaudatus.

C-type lectins are carbohydrate-binding proteins that play important roles in immunity by serving as pattern recognition receptors. In the present study, a novel nattectin-like C-type lectin was obtained from the weather loach, Misgurnus anguillicaudatus, designated as MaCTL. MaCTL encodes a peptide with 165 amino acids, with a signal peptide and a single C-type lectin domain (CTLD), containing a galactose-specific QPD motif and a conserved Ca2+ -binding site. Transcripts of MaCTL were significantly upregulated after immune challenge with its pathogen A. hydrophila. In vitro assays with recombinant MaCTL protein revealed that it exhibited hemagglutinating and bacterial agglutinating activities, in a Ca2+ -dependent manner. MaCTL was found to bind to a wide range of bacteria, as well as bind to bacterial polysaccharides LPS and PGN. Moreover, MaCTL displayed antimicrobial activity by inhibiting the growth of bacteria. These results collectively suggest that MaCTL is involved in the antibacterial defence of weather loach.

A toll receptor is involved in antibacterial defense in the oriental river prawn, Macrobrachium nipponense.

Toll-like receptors (TLRs) play an important role in the activation of innate immune response in animals. In this study, we identified a TLR from the oriental river prawn, Macrobrachium nipponense (MnToll1) and investigated its functions in immunity. The MnToll1 protein shares similar structural characteristics with other known Toll family proteins. MnToll1 transcripts are broadly distributed in all of the examined tissues, and its expression level was significantly up-regulated by bacterial challenge. RNAi-mediated knockdown of MnToll1 significantly impaired the survivability of Vibrio-challenged prawns. RNAi experiments also revealed that the expression of several antimicrobial peptide genes were regulated by MnToll1. Moreover, we found the extracellular region of MnToll1 could directly bind to bacteria and bacterial glycoconjugates. These findings suggest that MnToll1 function as a pattern recognition receptor to recognize invading pathogen and initiate downstream gene expression, to participate in antibacterial defense of M. nipponense.

A mannose receptor is involved in the anti-Vibrio defense of red swamp crayfish.

Mannose receptor (MR), a member of pattern-recognition receptors (PRRs), is the first MR family member to be discovered that plays a critical role in immunity. The function of MRs has been reported in mammals and teleosts while none in invertebrates. In the present study, we identified a MR-like gene (designated as PcMR) from red swamp crayfish, Procambarus clarkii. The PcMR cDNA is 6848 bp long with a 6288 bp open reading frame that encodes a polypeptide with 2095 amino acid residues. PcMR transcripts were mainly detected in hepatopancreas and hemocytes, and upregulated by Vibrio anguillarum challenge. The PcMR protein contained 14 C-type lectin domains (CTLDs) and they were divided into four fragments (CTLD 1-3, CTLD 4-6, CTLD 7-10, CTLD 11-14). The four recombinant proteins encoded by the four fragments were all expressed and purified. Microorganism-binding and sugar-binding assay showed that CTLD 1-3, CTLD 4-6, CTLD 7-10, CTLD 11-14 could bind to a variety of bacteria, as well as glycoconjugates on the bacterial surface. Moreover, they agglutinated bacteria in a calcium-dependent manner. Bacteria clearance experiment manifested that the mixed proteins facilitated the clearance of injected bacteria in crayfish. PcMR silencing by siRNA interference impaired the bacterial clearance ability. These results suggest PcMR is involved in the antibacterial defense of crayfish, and this study will help us better understand the functions of invertebrate MRs.

Characterization of an immune deficiency (IMD) homolog from the oriental river prawn, Macrobrachium nipponense.

The immune deficiency (IMD) signal pathway mediates innate immunity against Gram-negative bacteria in crustaceans. In the present study, an IMD homolog (MnIMD) from the oriental river prawn, Macrobrachium nipponense was identified. The full-length cDNA of MnIMD was 782bp with an open reading frame of 549 bp that encodes a putative protein of 182 amino acids including a death domain at the C-terminus. Multiple alignment analysis showed that IMDs in prawn M. nipponense and other crustaceans shared high similarity. The recombinant protein of MnIMD was expressed and purified for further functional analyses. Western blot analysis indicated that MnIMD was present in many tissues, but with the highest level in the gills, which was consistent with the qRT-PCR results. After Vibrio parahaemolyticus challenge, MnIMD was significantly induced in gills. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of different antimicrobial peptide (AMP) genes, including Cru4 and Cru6. These results are helpful in promoting research on the innate immunity in M. nipponense.