Immune activation by a multigene family of lectins with variable tandem repeats in oriental river prawn (Macrobrachium nipponense).

Genomic regions with repeated sequences are unstable and prone to rapid DNA diversification. However, the role of tandem repeats within the coding region is not fully characterized. Here, we have identified a new hypervariable C-type lectin gene family with different numbers of tandem repeats (Rlecs; R means repeat) in oriental river prawn (Macrobrachium nipponense). Two types of repeat units (33 or 30 bp) are identified in the second exon, and the number of repeat units vary from 1 to 9. Rlecs can be classified into 15 types through phylogenetic analysis. The amino acid sequences in the same type of Rlec are highly conservative outside the repeat regions. The main differences among the Rlec types are evident in exon 5. A variable number of tandem repeats in Rlecs may be produced by slip mispairing during gene replication. Alternative splicing contributes to the multiplicity of forms in this lectin gene family, and different types of Rlecs vary in terms of tissue distribution, expression quantity and response to bacterial challenge. These variations suggest that Rlecs have functional diversity. The results of experiments on sugar binding, microbial inhibition and clearance, regulation of antimicrobial peptide gene expression and prophenoloxidase activation indicate that the function of Rlecs with the motif of YRSKDD in innate immunity is enhanced when the number of tandem repeats increases. Our results suggest that Rlecs undergo gene expansion through gene duplication and alternative splicing, which ultimately leads to functional diversity.

Taiman negatively regulates the expression of antimicrobial peptides by promoting the transcription of cactus in Macrobrachium nipponense.

In insects, Taiman (Tai) participates in the juvenile hormone, 20-hydroxyecdysone, insulin, and Hippo signaling pathways. However, the role of Tai in crustacean innate immunity is less known. In this study, four Tai isoforms (MnTai-A, MnTai-B, MnTai-C, and MnTai-D) produced by alternative splicing were identified from Macrobrachium nipponense. The obtained genome sequences indicated that MnTai DNA has more than 20 exons and 19 introns. The second to last (-exon2) and the third to last (-exon3) exons can be alternatively spliced. The loss of -exon2 or -exon3 produces MnTai-B or MnTai-C, respectively. Both exons are absent in MnTai-D. The full-length cDNA of MnTai-A (including all exons) was 6894 bp with an open reading frame of 4998 bp that encoded a protein of 1665 amino acids. MnTaiA contains the conservative structure of the Tai family and clustered with nuclear receptor coactivator from shrimp. All these four isoforms were widely distributed in a variety of tissues with the highest expression level in the hepatopancreas except MnTaiC. The transcriptional levels of total Tai genes (designated as MnTaiT) in the hepatopancreas and gills were regulated by bacterial or viral challenge. Knockdown of MnTaiT increased the expression of anti-microbial peptides (AMPs) during Vibrio parahaemolyticus infection. Further study indicated that the negative regulation of AMP gene expression by prawn Tai was mediated through its positive regulation of cactus. Our research provides valuable information that prawn Tai isoforms are involved in innate immunity.

Function of the MOB kinase activator-like 1 in the innate immune defense of the oriental river prawn (Macrobrachium nipponense).

The monopolar spindle one binder (MOB) protein, a key signal transducer of the Hippo signaling pathway, is involved in growth control and cancer. In this study, a new MOB kinase activator-like 1 of the oriental river prawns, Macrobrachium nipponense, (MnMOB1) was isolated and characterized. The open reading frame of MnMOB1 consisted of 651 nucleotides that encoded 216 amino acid residues and contained the Mob1_phocein domain. Phylogenetic analysis revealed that MnMOB1 clustered together with the MOB1 from Penaeus vannamei. The distribution of MnMOB1 expression in various tissues of normal prawn revealed that the MnMOB1 expression was highest in the hepatopancreas followed by those in the intestines, gill, heart, stomach, and hemocytes. In prawns challenged with Staphylococcus aureus and Vibrio parahaemolyticus, the expression levels of MnMOB1 in the hepatopancreas, gills, and intestine were upregulated. Furthermore, the expression levels of crustins and anti-lipopolysaccharide factors in prawn injected with S. aureus and V. parahaemolyticus and MnMOB1 knockdown were significantly decreased relative to those in the control group. These findings indicated that MnMOB1 is involved in the regulation of antimicrobial peptide expression and plays a crucial role in the innate immunity of M. nipponense.

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.

Novel fibrinogen-related protein with single FReD contributes to the innate immunity of Macrobrachium rosenbergii.

Fibrinogen-related proteins (FREPs) are widely found in vertebrates and invertebrates, and they play crucial roles in innate immunity. Here, a new FREP named as MrFREP was identified from giant freshwater prawn (Macrobrachium rosenbergii). The full-length cDNA of MrFREP measures 1649 bp in length and consists of a 1086 bp open reading frame encoding a polypeptide composed of 361 amino acids. The MrFREP sequence has a signal peptide with 20 amino acids and a fibrinogen-related domain (FReD) with 223 amino acids. Phylogenetic analysis showed that MrFREP was grouped with FREPs from Marsupenaeus japonicus and Litopenaeus vannamei. BLASTp results showed that it had 43% identity with a FREP from M. japonicus. The expression of MrFREP was higher in gills, intestine, and hepatopancreas than in hemocytes, heart, stomach, and muscles. The expression levels of MrFREP in gills and intestine were obviously upregulated after they were exposed to Vibrio parahaemolyticus or White spot syndrome virus infection. Recombinant MrFReD protein (rMrFReD) could bind to Gram-positive and Gram-negative bacteria and agglutinate the tested bacteria in the presence of calcium. rMrFReD demonstrated lipopolysaccharide and peptidoglycan binding activities. rMrFReD could accelerate the clearance of V. parahaemolyticus in vivo. These results suggested that MrFREP could function as a pattern recognition receptor contributing to the innate immunity of M. rosenbergii.

Function of two ficolin-like proteins in innate immune defense of the oriental river prawn, Macrobrachium nipponense.

Ficolins have crucial functions in recognizing and eliminating pathogens in innate immunity. In this study, we identified two ficolin-like genes from the oriental river prawn, Macrobrachium nipponense. These genes were designated as MnFico1 and MnFico2. MnFico1 cDNA has 1600 bp, whereas MnFico2 has 1486 bp. In addition to a coiled-coil region or a low complexity region, the two ficolins both contained a signal peptide and a fibrinogen-related domain. qRT-PCR results showed that the highest expression level of MnFico1 expression was in the gills, whereas that of MnFico2 was in the heart. The transcripts of MnFico1 and MnFico2 could both respond to bacteria challenge. The transcription of antilipopolysaccharide factors ALFs (MnALF1 and MnALF2) and crustin genes (MnCru4, MnCru5, MnCru6, and MnCru7) was inhibited in the gills of MnFico1 or MnFico2 knockdown prawns at 24 h Vibrio parahaemolyticus challenge. Recombinant proteins of rMnFico1 and rMnFico2 could bind toward diverse bacteria and agglutinate Gram-negative and Gram-positive bacteria with the presence of calcium (Ca2+). rMnFico1 and rMnFico2 proteins also have lipopolysaccharide and peptidoglycan binding activity. Both recombinant ficolin proteins could help the prawn to facilitate the clearance of V. parahaemolyticus in vivo. Our results suggested that MnFico1 and MnFico2 might serve as pattern recognition receptors in M. nipponense.

Single CRD containing lectin from Macrobrachium rosenbergii (MrLec) participates in innate immunity against pathogen infections.

As a type of pattern-recognition proteins, lectins perform important functions in the innate immunity of crustaceans, including prawns. Although several reports showed that C-type lectin domain family (CLEC) importantly functions in host-pathogen interactions, limited research has focused on CLEC in Macrobrachium rosenbergii. In the present study, a new single CRD containing CLEC (designated as MrLec) was reported in freshwater prawns, M. rosenbergii. The full-length cDNA of MrLec consisted of 1027 bp with an open reading frame of 801 bp, which encoded a peptide of 266 amino acid residues. Genomic sequence for MrLec was also obtained from the M. rosenbergii, which contain 4 exons and 3 introns. MrLec was found to contain a single carbohydrate-recognition domain with an EPN motif. MrLec was ubiquitously distributed in various tissues of a normal prawn, particularly in the hepatopancreas and gills. MrLec expression in the gills was significantly upregulated after a challenge with Vibrio parahaemolyticus and downregulated at 24 h after MrLec RNA interference (MrLec-RNAi). The expression levels of some AMPs, including antilipopolysaccharide factor 1 (Alf1) and lysozyme 2 (Lyso2), also markedly decreased after MrLec-RNAi. Recombinant MrLec can agglutinate (Ca(2+)-dependent) and bind both Gram-negative and Gram-positive bacteria. Results suggested that MrLec participates in the recognition of invading pathogens and functions in the immune response of prawn against pathogen infections.

Involvement of Relish gene from Macrobrachium rosenbergii in the expression of anti-microbial peptides.

Relish is an NF-kB transcription factor involved in immune-deficiency (IMD) signal pathway. In this study, a Relish gene (MrRelish) was identified from Macrobrachium rosenbergii. The full length of MrRelish comprises 5072 bp, including a 3510 bp open reading frame encoding a 1169 bp amino acid protein. MrRelish contains a Rel homology domain (RHD), a nucleus localization signal, an IκB-like domain (6 ankyrin repeats), and a death domain. Phylogenetic analysis showed that MrRelish and other Relish from crustaceans belong to one group. MrRelish was expressed in all detected tissues, with the highest expression level in hemocytes and intestines. MrRelish was also upregulated in hepatopancreas at 6 h after Vibrio anguillarum challenge. The over-expression of MrRelish could induce the expression of antimicrobial peptides (AMPs), such as Drosophila Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), and Cecropin (CecA) and shrimp Penaeidin (Pen4). The RNAi of MrRelish in gills showed that the expression of crustin (cru) 2, Cru5, Cru8, lysozyme (Lyso) 1, and Lyso2 was inhibited. However, the expression of anti-lipopolysaccharide factor (ALF) 1 and ALF3 did not change when MrRelish was knocked down. These results indicate that MrRelish may play an important role in innate immune defense against V. anguillarum in M. rosenbergii.

Identification and molecular characterization of a peritrophin-like gene, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis.

Peritrophin was first isolated from insect peritrophic membrane (PM) and was thought to protect insects from invasion of microorganisms and to stimulate digestion of food. In this study, a peritrophin-like gene (EsPT) was obtained from Eriocheir sinensis. The full length cDNA of EsPT was 1232 bp, which contained 1005 bp ORF encoding a protein of 334 amino acids, including a 22 amino acid signal peptide, and 3 conserved chitin binding type 2 domains (ChtBD2) characterized by having a 6-cysteine motif. Phylogenetic analysis showed that EsPT was clustered together with 2 insect peritrophin-44-like proteins (MdP44L from Musca domestica and CcP44L from Ceratitis capitata), an insect chitin binding peritrophin-A domain containing protein (CfPT from Coptotermes formosanus) and a crustacean peritrophin (MnPT from Macrobrachium nipponense). Tissue distribution analysis revealed that EsPT was mainly expressed in hepatopancreas, intestine and hemocytes. The expression of EsPT is regulated by lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus and Aeromonas hydrophila challenge. The recombinant EsPT could bind to different microbes, and enhanced the clearance of V. parahaemolyticus in vivo. In crabs, silencing of EsPT by siRNA suppressed the elimination of V. parahaemolyticus and increasing number of bacteria, finally upregulated the expression of anti-lipopolysaccharide factor (ALF) and clip domain serine proteases (cSP). The results might indicate that EsPT was involved in the anti-bacterial innate immunity of crabs.