Expression and functional characterization of the CUB domain-containing protein from the triangle sail mussel (Hyriopsis cumingii) in response to pathogenic infection.

The complement C1r/C1s, Uegf, and Bmp1 (CUB) domains, which are most exclusively found in extracellular and plasma membrane-related proteins, are involved in various biological processes. In this study, a CUB domain-containing protein (designed as HcCDCP) was cloned and characterized from freshwater pearl mussel (Hyriopsis cumingii). The 2280 bp complete cDNA of the HcCDCP contained a 1002 bp open reading frame, which encoded a protein with 333 amino acids. The predicted HcCDCP protein contained a typical CUB domain and a transmembrane region. The tissue distribution analysis indicated that the HcCDCP was detected in all tissues, and the highest expression was found in hepatopancreas followed by gills. After infection with bacteria (i.e., Staphylococcus aureus and Vibrio parahaemolyticus), virus (white spot syndrome virus) and virus analogs (poly[I:C]), the mRNA level of the HcCDCP was significantly upregulated, suggesting that the HcCDCP might be involved in host immune defense response. The RNA interference revealed that the silencing of the HcCDCP could evidently inhibit the expression levels of lysozyme and tumor necrosis factor. Moreover, the recombinant protein of the CUB domain (rCUB) possessed binding capacity to eight different kinds of bacteria. The polysaccharide binding assay showed that the rCUB specifically bound to lipopolysaccharide, peptidoglycan, and D-mannose. This study provided valuable information for exploring the biological roles of CDCPs in the host defense system of mollusks.

Identification of a C-type lectin possessing both antibacterial and antiviral activities from red swamp crayfish.

C-type lectins are important immune molecules that participate in crustacean defense response. The present work reports a novel C-type lectin (PcLec6) from the red swamp crayfish Procambarus clarkii. PcLec6 encodes a single-peptide protein of 385 amino acids, which include a C-type lectin domain (CTLD) and a serine-rich region. PcLec6 expression in lymph organ and gills was up-regulated after bacterial challenge by Vibrio alginolyticus or white spot syndrome virus (WSSV). Recombinant full-length PcLec6 or its CTLD proteins were used for the functional analyses. Results showed that these two proteins had the capacity to bind to carbohydrates and bacteria. Both the full-length PcLec6 and CTLD facilitated the bacterial clearance, but only full-length PcLec6 protected crayfish from WSSV infection. Furthermore, PcLec6 regulated the expression of ALF genes. These results suggest that PcLec6 is involved in the innate immune response of crayfish against both bacterial and viral pathogens.

Characterization of a double WAP domain-containing protein from the red swamp crayfish Procambarus clarkii.

Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 amino acids consisting of signal peptide of 18 residues, and two tandem WAP domains of 38 and 44 residues. Multiple alignment indicates the presence of conserved motifs in both WAP domains, and phylogenetic analysis shows that Pc-DWD is a new member of the type-IV crustin family. Pc-DWD transcripts were found most abundantly in hemocytes, gills, intestine and heart, and induced by Vibrio anguillarum, Staphylococcus aureus and white spot syndrome virus challenge. RNAi knockdown of Pc-DWD expression led to increased expression of white spot syndrome virus genes and increased crayfish mortality after virus infection. Recombinant Pc-DWD exhibited strong protease inhibitory activity towards commercial subtilicin A and protease K. Pc-DWD inhibited the crude proteases from V. anguillarum and S. aureus cultures and from the crayfish tissue extracts. We infer that Pc-DWD acts in crayfish bacterial and viral immunity.

HcToll3 was involved in anti-Vibrio defense in freshwater pearl mussel, Hyriopsis cumingii.

Toll-like receptors (TLRs) play an important role in the activation of innate immune response but their functions in bivalves remain largely unknown. In this study, we identified a TLR from the freshwater pearl mussel Hyriopsis cumingii (HcToll3) and investigated its functions in immunity. The full-length cDNA of HcToll3 is 3852 bp and includes an open reading frame (ORF) of 3228 bp that encodes a polypeptide of 1075 amino acids. The predicted HcToll3 protein shares similar structural characteristics with other known Toll family proteins. Quantitative real-time PCR analysis revealed that HcToll3 mRNA is broadly expressed in all of the examined tissues; its transcript level was significantly up-regulated by challenge with gram-negative bacteria Vibrio parahaemolyticus or lipopolysaccharide, but not gram-positive Staphylococcus aureus or peptidoglycan. RNA interference by siRNA results showed that HcToll3 regulated expression of whey acidic protein (HcWAP) and lysozymes (HcLyso1 and HcLyso2) in vivo and knockdown of HcToll3 suppressed the elimination of V. parahaemolyticus. These findings suggest that HcToll3 might be involved in anti-Vibrio defense in H. cumingii.

Three STATs are involved in the regulation of the expression of antimicrobial peptides in the triangle sail mussel, Hyriopsis cumingii.

Janus kinase (Jak) and signal transducers and activators of transcription (STAT) signaling pathway is associated in antiviral and antibacterial immune response. Previous studies primarily investigated the function of STATs in mammals. For most invertebrates, only one STAT was found in each species, such as STAT92E was found in Drosophila melanogaster. The studies, which focus on the functional difference between various STATs in the same species of invertebrate, are limited. In the present study, three STATs (HcSTAT1, HcSTAT2 and HcSTAT3) were identified in triangle shell pearl mussel, Hyriopsis cumingii. Phylogenetic analysis showed that HcSTAT1 and HcSTAT3 were clustered with Homo sapiens STAT5, and HcSTAT2 was clustered with Pinctada fucata STAT and Crassostea gigas STAT6. All three STATs could be detected in all tested tissues (hemocytes, hepatopancreas, gill, mantle and foot), and were induced expression when challenged with Staphylococcus aureus or Aeromonas hydrophilia in hemocytes and hepatopancreas. HcSTAT1 regulated the expression of HcDef, HcWAP, HcThe and HcTNF. The expression of HcWAP and HcTNF was down-regulated in HcSTAT2-RNAi mussel. And HcSTAT3 affected the expression of HcTNF. The study is the first report of different functions in antibacterial immune responses between STATs in mollusks.

Newly identified PcToll4 regulates antimicrobial peptide expression in intestine of red swamp crayfish Procambarus clarkii.

Tolls or Toll-like receptors (TLRs) have an essential role in initiating innate immune responses against pathogens. In this study, a novel Toll gene, PcToll4, was first identified from the intestinal transcriptome of the freshwater crayfish, Procambarus clarkii. The PcToll4 cDNA is 4849bp long with a 3036bp open reading frame that encodes a 1011-amino acid protein. PcToll4 contains a signal peptide, 13 LRR domains, 3 LRR TYP domains, 2 LRR CT domains, an LRR NT domain, a transmembrane region, and a TIR domain. Quantitative RT-PCR analysis revealed that PcToll4 mRNA was detected in all tested tissues, and the expression of PcToll4 in the intestine was significantly upregulated after white spot syndrome virus (WSSV) challenge. Overexpression of PcToll4 in Drosophila Schneider 2 (S2) cells activates the antimicrobial peptides (AMPs) of Drosophila, including metchnikowin, drosomycin, attacin A, and shrimp Penaeidin-4. Results of RNA interference by siRNA also showed that PcToll4 regulates the expressions of 5 anti-lipopolysaccharide factors (ALFs) in the intestine of crayfish. Our findings suggest that PcToll4 is important for the innate immune responses of P. clarkii because this gene regulates the expressions of AMPs against WSSV.

Characterization of a gC1qR from the giant freshwater prawn, Macrobrachium rosenbergii.

gC1qR, as a multicompartmental and a multifunctional protein, plays an important role in innate immunity. In this study, a gC1qR homolog (MrgC1qR) in the giant freshwater prawn, Macrobrachium rosenbergii was identified. MrgC1qR, a 258-amino-acid polypeptide, shares high identities with gC1qR from other species. MrgC1qR gene was expressed in different tissues and was highest expressed in the hepatopancreas. In addition, the MrgC1qR transcript was significantly enhanced after 6 h of white spot syndrome virus (WSSV) infection or post 2 h, 24 h of Vibrio anguillarum challenge compared to appropriate controls. Moreover, recombinant MrgC1qR (rMrgC1qR) had bacterial binding activity, the result also revealed that rMrgC1qR could bind pathogen-associated molecular patterns (PAMPs) such as LPS or PGN, suggesting that MrgC1qRmight function as a pathogen-recognition receptor (PRR). Furthermore, glutathione S-transferase (GST) pull-down assays showed that rMrgC1qR with GST-tag could bind to rMrFicolin1 or rMrFicolin2 with His-tag. Altogether, these results may demonstrate a role for MrgC1qR in innate immunity in the giant freshwater prawns.

A novel Toll like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii.

Animal Toll-like receptors (TLRs) are involved in innate immunity. Toll proteins are generally transmembrane proteins. In this study, an atypical Toll-like receptor (HcToll-2) was identified from the triangle-shell pearl mussel Hyriopsis cumingii, which belongs to phylum Mollusca. Unlike the typical Toll like receptors with extracellular leucine-rich repeats (LRRs), transmembrane, and intracellular Toll/interleukin-1 receptor (TIR) domains, HcToll-2 has two homologous TIR domains located at the C-terminal (designated as HcTIR1 and HcTIR2) and lacks a transmembrane domain. Phylogenetic analysis showed that HcTIR1 was clustered with TIR of sea anemone Toll, and HcTIR2 was clustered with TIR of Drosophila Toll. HcToll-2 mRNA could be detected in the hepatopancreas and was upregulated after challenge with Escherichia coli and Staphylococcus aureus. Recombinant HcLRR protein with GST tag could bind to bacteria and also to LPS and PGN. Over-expression of both HcTIR1 and HcTIR2 induced drosomycin genes in Drosophila S2 cells. RNAi analysis showed that HcToll-2 was required for the expression of theromacin, which is a cysteine-rich antimicrobial peptide (AMP) gene. This research is the first report of an atypical Toll-like receptor HcToll-2 involved in antibacterial immunity through induction of AMP expression.

Function of two novel single-CRD containing C-type lectins in innate immunity from Eriocheir sinensis.

C-type lectin is one of the pattern-recognition proteins of the non-self-innate immune system in invertebrates. In this study, two novel C-type lectin cDNAs (EsCTL1 and EsCTL2) of Eriocheir sinensis were cloned and characterized. EsCTL1 has 169 amino acids, whereas EsCTL2 has 164 amino acids. These two lectins contain one carbohydrate-recognition domain. Phylogenetic analysis showed that EsCTL1 and EsCTL2 were not clustered with other reported lectins from crabs. EsCTL1 and EsCTL2 were expressed only in the hepatopancreas, as detected by real-time PCR. When healthy crabs were challenged with lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, or Aeromonas hydrophila, the expression levels of EsCTL1 and EsCTL2 were significantly regulated. The recombinant EsCTL1 and EsCTL2 can agglutinate both Gram-positive (S. aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and A. hydrophila) in a Ca2+ -dependent manner. The recombinant EsCTL1 and EsCTL2 can directly bind to LPS and PGN and to all tested microorganisms (S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila). Furthermore, rEsCTL1 and rEsCTL2 may facilitate the clearance of V. parahaemolyticus in vivo. These results suggest that EsCTL1 and EsCTL2 may have important roles in the anti-bacterial immunity of Chinese mitten crab.

Cloning and characterization of two different ficolins from the giant freshwater prawn Macrobrachium rosenbergii.

Ficolins, a kind of lectin containing collagen-like and fibrinogen-related domains (FReDs, also known as FBG or FREP), are involved in the first line of host defense against pathogens. In this study, two ficolins, namely, MrFico1 and MrFico2, from the giant freshwater prawn Macrobrachium rosenbergii were identified. In contrast to other ficolins, these two ficolins have no collagen-like domain, but such ficolins contain a coiled region and a FReD domain. Phylogenetic analysis showed that MrFico1 and MrFico2, together with two ficolin-like proteins from Pacifastacus leniusculus, belonged to one group. Quantitative RT-PCR (qRT-PCR) showed that both MrFico1 and MrFico2 were expressed in hepatopancreas, stomach and intestine, with the highest expression in stomach for MrFico1, compared to the highest expression in hepatopancreas for MrFico2. qRT-PCR analysis also showed that MrFico1 was obviously upregulated upon Vibrio anguillarium challenge, while MrFico2 was upregulated after challenged by V. anguillarium or white spot syndrome virus. Bacterium-binding experiment showed that MrFico1 and MrFico2 could bind to different microbes, and sugar-binding assay revealed that these two ficolins could also bind to lipopolysaccharide and peptidoglycan, the glycoconjugates of bacteria surface. Moreover, these two ficolins could agglutinate bacteria in a calcium-dependent manner, and the results of bacteria clearance experiment showed that both ficolins could facilitate the clearance of injected bacteria in the prawn. Our results suggested that MrFico1 and MrFico2 may function as pattern-recognition receptors in the immune system of M. rosenbergii.

An LDLa domain-containing C-type lectin is involved in the innate immunity of Eriocheir sinensis.

C-type lectins (CTLs) have crucial functions in recognizing and eliminating pathogens in innate immunity. This study identified a novel low-density lipoprotein receptor class A (LDLa) domain-containing CTL, designated as EsCTLDcp, from the Chinese mitten crab Eriocheir sinensis. The EsCTLDcp cDNA is 1258 bp long, with a 975 bp open reading frame that encodes a 324-amino acid protein. EsCTLDcp contains a signal peptide, an LDLa, and a single C-type lectin-like domain. EsCTLDcp was only expressed in the hepatopancreas of normal crabs, and its expression was regulated following crab challenge with pathogen-associated molecular patterns and with bacteria. The recombinant EsCTLDcp agglutinates Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. rEsCTLDcp also binds to various bacteria including S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila. The rEsCTLDcp protein helped the crabs clear the virulent Gram-negative bacterium V. parahaemolyticus in vivo, as well as interacted with VP24, an envelope protein of white spot syndrome virus (WSSV). These data suggest that EsCTLDcp functions as a pattern-recognition receptor involved in the innate immunity of E. sinensis.

Cloning and characterization of a clip domain serine protease and its homolog (masquerade) from Eriocheir sinensis.

Serine proteinases (SPs) or SP homologs (SPHs) including clip domain SPs (cSPs) or SPHs (cSPHs) play critical roles in digestion, embryonic development, hemolymph coagulation, and melanization. In this study, one cSP (EscSP) and one SPH, similar to Drosophila masquerade (EsMas), were identified from hepatopancreas of the Chinese mittern crab Eriocheir sinensis. They both possess the clip domains at the N-terminal, EscSP has only one clip domain, but EsMas has seven clip domains. One SP or SP-like domain was at the C-terminal of EscSP and EsMas respectively. In contrast to EscSP, absence of a catalytic residue of Ser resulted in the loss of SP activity of EsMas. Tissue distribution analysis showed that EscSP mRNA was mainly expressed in hepatopancreas, nerve and eyestalk tissue; whereas the EsMas transcript was mainly distributed in eyestalk, muscle, nerve and hemocytes. EscSP in hemocytes showed significant increase after a lipopolysaccharide (LPS) or peptidoglycan (PGN) challenge. However, down-regulation of EsMas was observed in hemocytes challenged by LPS from 2 to 24 h, by contrast EsMas could be induced by PGN challenge at 2 and 24 h. All these findings indicated that EscSP and EsMas might be involved in the innate immune defenses in E. sinensis.

Cloning and identification of four Mu-type glutathione S-transferases from the giant freshwater prawn Macrobrachium rosenbergii.

Glutathione S-transferases (GSTs) are essential components of the cellular detoxification system because of their capability to protect organisms against the toxicity of reactive oxygen species (ROSs). Four different GSTs (MrMuGST1-MrMuGST4) showing similarities with Mu-type GSTs were cloned from the hepatopancreas of Macrobrachium rosenbergii. These four GSTs have 219, 216, 218 and 219 amino acids in length, respectively. MrMuGST1-MrMuGST4 proteins all have a G-site in the N-terminus and an H-site in the C-terminus. Phylogenetic analysis reveals that four Mu-type GSTs are classified into two different clades (MrMuGST2 one clade; MrMuGST1, MrMuGST3 and MrMuGST4 other clades). Nonetheless, no site under positive selection was detected but rapid evolution was found in the few of MuGST genes. Reverse transcription-polymerase chain reaction (RT-PCR) results showed that MrMuGST1 and MrMuGST2 transcripts were expressed in all detected tissues, however, MrMuGST3 and MrMuGST4 were just mainly expressed in hepatopancreas and intestines. Quantitative RT-PCR analysis showed that MrMuGST1 and MrMuGST2 were down-regulated upon Vibrio anguillarum challenge, whereas MrMuGST3 and MrMuGST4 were quickly up-regulated 2 h after the Vibrio challenge. Our results imply that different Mu-type GSTs may respond to Vibrio challenge with different manners.

Diversity of lectins in Macrobrachium rosenbergii and their expression patterns under spiroplasma MR-1008 stimulation.

Lectins play important roles in crustacean innate immunity through recognition of foreign pathogens. In this study, 20 lectins including C-type lectins [dual-carbohydrate recognition domain (CRD) type and single-CRD type], L-type lectin, and lectin with low-density lipoprotein class A (LDLa) domain were identified from the freshwater prawn Macrobrachium rosenbergii. The tissue distribution and expression patterns of these lectins under spiroplasma strain MR-1008 challenge were investigated. Most of the lectins were found to be mainly distributed in the hepatopancreas. Lectin5, Lectin14, Lectin17, and Lectin18 exhibited the highest expression level in the hemocytes, nerve, intestine, and heart, respectively. MrLec1 to MrLec6 (dual-CRD lectins) in the hepatopancreas were up-regulated by spiroplasma challenge. Single-CRD lectins reached the highest level at 72 h after spiroplasma challenge. Lectin9 and Lectin15 both belong to L-type lectins. At post-spiroplasma challenge, Lectin9 expression was up-regulated, whereas Lectin15 expression was down-regulated. Lectin11 with LDLa domain showed the highest level after 12 h Lectin18 and Lectin20, namely, CD209, were also up-regulated by spiroplasma challenge. Lectin14, a C-type lectin, quickly reached the highest level after 2 h Lectin16 showed the highest level after 72 h Lectin5 reached the highest level in cultured hemocytes after 6 h Lectin17 in the intestine and Lectin14 in the nerve were slightly up-regulated after 6 and 2 h, respectively. Our research results indicate that lectins may play important roles in early or late immune responses against spiroplasma challenge.

The first Toll receptor from the triangle-shell pearl mussel Hyriopsis cumingii.

Toll receptor was first discovered in Drosophila and has an important function in the innate immunity of invertebrates. In this study, the Toll receptor HcToll1 from Hyriopsis cumingii with a full length of 3810 bp consisting of a 3687 bp ORF that encodes a total of 1228 amino acids protein was selected for further study. The HcToll1 protein consisted of a signal peptide, 17 LRR domains, 2 LRRCT domains, 1 LRRNT domain, 1 TM domain, and 1 TIR domain. Phylogenetic analysis results showed that HcToll1 was clustered in one group together with other mollusca tolls. RT-PCR analysis results showed that HcToll1 was expressed in all tested tissues such as hemocytes, hepatopancreas, gills, and mantle. qRT-PCR analysis results showed that HcToll1 expression was increased by the presence of Escherichia coli, Vibrio anguillarum, Staphyloccocus aureus, and White Spot Syndrome Virus (WSSV). Over-expression of HcTIR could up-regulate expression of drosomycin gene in Drosophila S2 cells. The results of our study indicated that HcToll1 is a functional Toll and it has an important function in the generation of innate immune responses of H. cumingii against microbial challenge.

Four invertebrate-type lysozyme genes from triangle-shell pearl mussel (Hyriopsis cumingii).

Lysozymes in animals have three types, namely chicken-type, goose-type, and invertebrate-type (i-type) lysozymes and all these 3 types have been found in bivalve mollusks. The i-type lysozymes in mollusks are involved in digestion and innate immunity. In this study, four different lysozyme genes that belong to i-type were identified from Hyriopsis cumingii. The HcLyso1 to HcLyso4 genes encode proteins with 144, 144, 161, and 228 amino acids, respectively, and contain a destabilase domain. HcLyso4 also contains SH3b domain in addition to its destabilase domain. Multiple alignments showed that two catalytic residues of Glu and Asp which were necessary for enzyme activity were present in i-type lysozymes. Phylogenetic analysis using CDS sequences of i-type lysozymes showed that these lysozymes can be divided into mollusk and crustacean clades, and that HcLyso1 to HcLyso4 all belong to the mollusk clades. Although there was no positive selection predicted in i-type lysozymes, some branches suffered rapid evolution. HcLyso1 is mainly expressed in hepatopancreas and can be detected in hemocytes. HcLyso2 is primarily expressed in hepatopancreas and can be detected in hemocytes Whereas, HcLyso3 can be detected mainly in hemocytes, hepatopancreas, gills, and mantle. HcLyso4 is expressed in hemocytes and hepatopancreas. qRT-PCR analysis showed that HcLyso1 to HcLyso4 were all nearly down-regulated by Vibrio or Staphylococcus aureus challenge. Moreover, our research indicated that HcLyso1 to HcLyso4 might play a key role in the innate immunity of mussel.

Three different anti-lipopolysaccharide factors identified from giant freshwater prawn, Macrobrachium rosenbergii.

Anti-lipopolysaccharide factor (ALF) is a type of basic protein and an important antimicrobial peptide that can bind and neutralize lipopolysaccharides (LPS). This protein shows a broad spectrum of antimicrobial activity. In this study, three forms of ALF designated as MrALF5, MrALF6, and MrALF7 were identified from giant freshwater prawn, Macrobrachium rosenbergii. MrALF5, MrALF6, and MrALF7 genes encode 133, 121, and 120 amino acids of the corresponding proteins, respectively. All these ALF proteins contain LPS-binding domain with two conserved cysteine residues. The genomic sequences of MrALF5 and MrALF7 were amplified. The genomic structures of MrALF5 and MrALF7 comprise three exons interrupted by two introns. Phylogenetic analysis showed that MrALF5, MrALF6, and MrALF7 were clustered into clade II. Evolutionary analysis showed that ALF genes from M. rosenbergii may suffer a rapid evolution. MrALF5 was expressed mainly in the hepatopancreas, gills, and heart. MrALF6 was mainly distributed in the intestine and hepatopancreas. The highest expression level of MrALF7 was detected in the hepatopancreas. MrALF6, as well as MrALF7, was downregulated by Escherichia coli challenge, and all three ALF genes were upregulated by Vibrio or white spot syndrome virus challenge. MrALF6 was also upregulated by Staphylococcus aureus challenge. In summary, the three isoforms of ALF genes may participate in the innate immune response against bacteria and virus infecting the giant fresh water prawn.