Qian Ren

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp, Fenneropenaeus chinensis.

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.

A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis.

The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillussubtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens.

Characterization of an immune deficiency homolog (IMD) in shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkii).

The immune deficiency (IMD) signal pathway mediates immunity against Gram-negative bacteria in Drosophila. Recent studies show that the IMD pathway also involves in antiviral innate immune responses. The functions of the pathway in crustacean immunity are largely unknown. In this paper, two IMDs (FcIMD and PcIMD), one of the key elements of the IMD pathway, were identified from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Both proteins have a death domain located at the C-terminal. FcIMD was mainly expressed in the gills and stomach and PcIMD was mainly detected in the heart, hepatopancreas, and stomach. FcIMD peaked in hemocytes at 12 h after white spot syndrome virus (WSSV) challenge and it peaked in the gills at 6 h after WSSV challenge, but it was decreased at 2 h and kept the low level to 24 h in hemocytes and no obviously change in gill after Vibrio anguillarum challenge. PcIMD first decreased in hemocytes at 2 h and peaked at 12 h in hemocytes after V. anguillarum challenge. It was also upregulated in gill after bacterial challenge, peaked at 2 h, and decreased at 6 h, and then gradually increased at 12-24 h. PcIMD has no significant change in hemocytes and gill after WSSV challenge. Western blot analysis detected FcIMD protein in all tissues, and immunocytochemical analysis localized FcIMD in the cytoplasm of hemocytes. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of three kinds AMP genes, including crustins, anti-lipopolysaccharide factors and lysozymes, in shrimp and crayfish. They are Cru 1, Cru 2, ALF 1, ALF 2 and Lys 1 in crayfish, and Cru1, Cru 3, ALF 6, ALF 8, and Lys2 in shrimp. These results suggest that although IMD distribution and expression patterns have some differences, the IMD pathway may have conserved function for AMP regulation in shrimp and crayfish immunity against Gram-negative bacteria.

A thioredoxin response to the WSSV challenge on the Chinese white shrimp, Fenneropenaeus chinensis

Thioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5 untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105 amino acids protein, and a 399 bp 3 UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is a widely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, it went up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challenged with WSSV was down-regulated from 2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps.

An acyl-CoA-binding protein (FcACBP) and a fatty acid binding protein (FcFABP) respond to microbial infection in Chinese white shrimp, Fenneropenaeus chinensis.

Acyl-CoA-binding protein (ACBP) and fatty acid-binding protein (FABP) are involved in lipid metabolism. ACBP plays a key role in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, vesicular trafficking, and gene regulation. In our study, a 536 bp cDNA of ACBP (FcACBP) was cloned and identified as a widely distributed gene in the Chinese white shrimp, Fenneropenaeus chinensis. Its expression in intestine was upregulated in response to white spot syndrome virus (WSSV) or Vibrio anguillarum infection. The expression patterns were confirmed by Western blot analysis. FABPs, members of the lipid-binding protein superfamily, play an important role in lipid metabolism and also participate in vertebrate innate immunity. A cDNA of FABP (FcFABP) cloned from the hepatopancreas of the shrimp was 715 bp in size and encoded a 14 kDa protein. FcFABP appeared to be a basic fatty acid binding protein with a predicted isoelectric point of 9.16. It showed sequence similarity to both vertebrate and invertebrate FABPs. Phylogenetic analysis showed that FcFABP, together with LvFABP, were clustered into one group. FcFABP was detected mainly in the hepatopancreas and expression level increased after a challenge with WSSV. FcFABP was down-regulated by V. anguillarum challenge. The protein also had bacterial binding activity. These two lipid metabolism related proteins may play important roles in shrimp innate immunity.

Two cysteine proteinases respond to bacterial and WSSV challenge in Chinese white shrimp Fenneropenaeus chinensis.

The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis (FcCathL, FcLegu) were obtained. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. Time-course analysis of FcCathL showed that FcCathL was upregulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12 h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2 h to 6 h and then recovered from 12 h to 24 h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time.

Expression of four trypsin-like serine proteases from the Chinese shrimp, Fenneropenaeus chinensis, as regulated by pathogenic infection.

Four trypsin-like serine proteases, designated as Fctry1, Fctry2, Fctry3, and Fctry4, were obtained from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. Three trypsin-like serine proteases, specifically Fctry1, 2, and 3 were observed to have the conserved catalytic triad (H, D, and S). Regarding Fctry4, the catalytic triad S was substituted by F, and the mature peptide was found to be inactive. Further analysis for similarities indicated that Fctry1, 2 and 4 were 89-92% identical to trypsins from Pacific white shrimp (Litopenaeus vannamei); while Fctry3 was only 42% identical to trypsins from salmon louse (Lepeophtheirus salmonis). The genomic organizations of Fctry1, 2 and 4 are also quite different from Fctry3. So, Fctry3 may be a new member of the trypsin-like serine protease family. Moreover, a semi-quantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR was carried out to analyze the distribution profiles and expression patterns after they were subjected to a bacterial and viral challenge. The results showed that the four trypsin-like serine proteases were upregulated in the hepatopancreas of shrimp infected with the white spot syndrome virus (WSSV), and Fctry3 increased after a bacteria challenge. Therefore, these trypsin-like serine proteases might be involved in the innate defense reactions against different pathogens in Chinese shrimp.

Molecular characterization and expression analysis of a chicken-type lysozyme gene from housefly （Musca domestica）

Lysozymes can hydrolyze bacteria and play an important role in animal digestion and innate immunity. The cDNA of a chicken-type lysozyme gene (Mdlys) was cloned from housefly (Musca domestica). The 484 bp full-length cDNA contains a 426 bp open reading frame (ORF) that encodes MdLys of 141 amino acids. Phylogenetic analysis indicated that the MdLys was similar to chicken-type lysozymes. Spatio-temporal expression of Mdlys was analyzed by RT-PCR. The Mdlys transcript can be detected in both midgut and fat body and was expressed at a relatively lower level at the embryo stage. Mdlys mRNA was upregulated 2 h post bacterial challenge, maintained for 2 to 6 h, and slightly declined from 12 to 24 h post-injection. Western blot analysis showed that MdLys was highly expressed in midgut and was also detected in the hemolymph and fat body. MdLys expression was slightly increased in midgut after challenging with Escherichia coli or Staphylococcus aureus. Its expression was also slightly increased in the fat body after challenging with S. aureus, but no obvious change occurred after E. coli challenge. MdLys expression in the hemolymph was not affected by bacterial challenge. In the developmental stages, MdLys expression levels had no obvious change from the first instar to the pupae stage. There was also no variation under 24 h starvation stress. Recombinant MdLys displayed inhibitory activity against Gram-negative and Gram-positive bacteria. Together, these results suggest that MdLys may play an important role in the innate immunity of houseflies.

Identification of three different types of serine proteases (one SP and two SPHs) in Chinese white shrimp

Serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, embryonic development, blood coagulation, and immune defense responses. In this paper, we identify one SP and two SPHs, including a masquerade SPH (FcMas), a CUB domain containing SP (FcCUBSP), and a single domain containing SPH (FcSPH2) in Chinese white shrimp, Fenneropenaeus chinensis. FcMas has a Gly-rich region formed by three repeats of LGGQGGG, a clip domain and a C-terminal SP-like domain. Absence of Ser catalytic residue results in the loss of serine protease activity of FcMas, which then functions as an SPH. FcCUBSP has a signal peptide, followed by a CUB domain and an SP domain. FcSPH2 has a signal peptide and an SP-like domain. Loss of one catalytic residue (H) makes FcSPH2 catalytically inactive, which is considered an SPH. Phylogenetic analysis shows that FcMas and other SPHs from shrimp or insect are classified into one group. FcSPH2 is grouped in the chymotrypsin family. RT-PCR results show that FcMas mRNA is mainly distributed in hemocytes and gills. FcCUBSP is only detected in gills, whereas FcSPH2 is found in hepatopancreas only. QRT-PCR is used to analyze changes of FcMas, FcCUBSP and FcSPH2 in some tissues challenged with white spot syndrome virus (WSSV) or Vibrio. FcMas in hemocytes is down-regulated by WSSV or Vibrio challenge, and down-regulated by WSSV in gills. However, it is up-regulated upon Vibrio challenge in gills. FcCUBSP in gills and FcSPH2 in hepatopancreas are up-regulated upon WSSV or Vibrio challenge. Results suggest the roles of FcMas, FcCUBSP and FcSPH2 in shrimps innate immunity.

Overexpression of a C-type lectin enhances bacterial resistance in red swamp crayfish Procambarus clarkii

C-type lectins play important roles in the innate immune system of crustaceans. In this study, a novel C-type lectin gene, designated as PcLec4, was obtained from the red swamp crayfish (Procambarus clarkii). Quantitative real-time polymerase chain reaction revealed that PcLec4 is mainly expressed in the crayfish hepatopancreas and intestine, and the PcLec4 mRNA expression is upregulated after challenged with the bacteria Vibrio anguillarum. PcLec4 was recombinantly expressed in Escherichia coli and anti-PcLec4 polyclonal antiserum was prepared. Binding experiments revealed that the recombinant PcLec4 binds to various bacteria and polysaccharides on the bacterial surface, which suggests that PcLec4 recognizes bacterial pathogens. Overexpression of PcLec4 in crayfish using the pIeLec4 vector was performed. The results show that the crayfish overexpressing PcLec4 eliminate injected V.xa0anguillarum more quickly than the control, which suggests that PcLec4 elicits further immune response for removing invading bacteria. The results of the survival experiment confirmed the function of PcLec4 in resisting V.xa0anguillarum because PcLec4 overexpression in crayfish significantly increased the crayfish survival rate. These results reveal that PcLec4 has an important role in the antibacterial immunity of crayfish, and inxa0vivo PcLec4 overexpression might be used as a disease control strategy in aquiculture.