Jiang-Feng Lan

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 novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosis

Crustins are cationic cysteine-rich antimicrobial peptides (AMPs) that contain multiple domains (glycine-rich, cysteine-rich, or proline-rich) at the N-terminus and whey acidic protein (WAP) domains at the C-terminus. Crustins have multiple functions, including protease inhibition and antimicrobial activity. Other functions of crustins need to be clarified. In this study, a novel crustin with a cysteine-rich region, and a single WAP domain, belonging to type I crustins, was identified in Marsupenaeus japonicus and designated as MjCru I-1. MjCru I-1 was expressed in various tissues. The expression of MjCru I-1 was upregulated in the hemocytes of shrimp challenged with bacteria. MjCru I-1 could bind to bacteria by binding to the cell wall molecules of the bacteria, such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoteichoic acid (LTA). The synthesized WAP domain of MjCru I-1 but not synthesized Cys-rich domain has antibacterial and agglutinative activities. Scanning electron microscope assay showed that the bacterial cells treated with sMjCru I-1 appeared to be disrupted and cracked compared with those of the control samples. The knockdown of MjCru I-1 could reduce bacterial clearance and injection of MjCru I-1 could significantly increase the survival rate of shrimp infected with Vibrio anguillarum and Staphylococcus aureus compared with those of the control samples. Further study discovered that MjCru I-1 could increase the hemocyte phagocytosis against V. anguillarum and S. aureus. These results suggest that MjCru I-1 has dual functions, bactericidal and phagocytosis promoting activities, in the antibacterial immunity of shrimp.

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish, Procambarus clarkii

ABSTRACT Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.

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.

β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity

The Toll signaling pathway plays an important role in the innate immunity of Drosophila melanogaster and mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense against Staphylococcus aureus by regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser276 that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease.

White spot disease caused by white spot syndrome virus (WSSV) leads to devastating losses in shrimp farming. The WSSV envelope protein VP28, can be used as subunit vaccines that can efficiently protect shrimp against WSSV disease. However, the function of the envelope protein VP19 was not confirmed, some researches found that VP19 could protect shrimp against WSSV, and other reports found it no any protection. To detect the functions of VP28 and VP19 and find a method to prevent this disease in red swamp crayfish Procambarus clarkii, we constructed the plasmid vectors pIevp28 and pIevp19, which contains the ie1 promoter and coding region of vp28 or vp19 of WSSV, respectively. The results of quantitative real-time PCR and western blot showed that the injected vectors could transcribe corresponding mRNAs and translate to the protein VP28 or VP19 in the crayfish. The vp28 or vp19 signal was detected on the third day post injection, and maintained its expression for 30days. The mortality of the crayfish with pIevp28 showed obvious decline compared with the controls (pIe and PBS injection). However, pIevp19 seems did not affect the mortality of the crayfish compared with the controls. Furthermore, only VP28 was found tightly bound to the host haemocytes under immunocytochemistry. The results suggest that the VP28 protein might protect shrimp from the virus through competitive inhibition. We also found that oral administration of Escherichia coli with pIevp28 could protect crayfish from white spot disease, but the E. coli with pIevp19 was not. Therefore, we think that oral administration of bacteria with pIevp28 is a potentially easy therapeutic way against white spot disease in aquaculture.

A fibrinogen-related protein (FREP) is involved in the antibacterial immunity of Marsupenaeus japonicus

Fibrinogen-related proteins (FREPs) in invertebrates have important functions in innate immunity. In this study, the cDNA of FREP was identified from the kuruma shrimp Marsupenaeus japonicus (MjFREP2). The full-length cDNA of MjFREP2 is 1138 bp with an open reading frame of 954 bp that encodes a 317-amino acid protein comprising a signal peptide and a fibrinogen-like domain. MjFREP2 could be detected in hemocytes, heart, hepatopancreas, gills, stomach, and intestines. MjFREP2 could also be upregulated in hemocytes after Vibrio anguillarum and Staphylococcus aureus challenge. Agglutination and binding assay results revealed that the recombinant MjFREP2 bound to bacteria and polysaccharides. Immunocytochemical analysis results showed that MjFREP2 proteins were mainly distributed in the cytoplasm of hemocytes from unchallenged shrimp and transported to the membrane or secreted out of the cell after V. anguillarum or S. aureus challenge. The secreted MjFREP2 bound to the bacteria presented in shrimp hemolymph. The overexpression of MjFREP2 could enhance bacterial clearance by inducing the phagocytosis of hemocytes. This ability was impaired by knockdown of MjFREP2 with RNA interference. The cumulative mortality of MjFREP2-silenced shrimp was significantly higher than that of the control shrimp. These results suggested that MjFREP2 has an important function in the antibacterial immunity of M. japonicus.

BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii.

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress-induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish.

A lysin MOTIF (LysM)-containing protein functions in antibacterial responses of red swamp crayfish, Procambarus clarkii

Lysin domain (LysM) is a widely spread domain in nature and could bind different peptidoglycans and chitin-like compounds in bacteria and eukaryotes. In plants, Lysin motif containing proteins are one of the major classes of pattern recognition proteins which can recognize GlcNAc-containing glycans and have important functions in plant immunity. However, their functions in animal immunity are still unclear. In this study, a cDNA encoding a LysM containing protein was identified from red swamp crayfish, Procambarus clarkii. The cDNA of PcLysM contained 1200 base pair nucleotides with an open reading frame of 702bp encoding a protein of 233 amino acid residues. The deduced protein had a calculated molecular mass of 25.950kDa and a pI of 6.84. Tissue distribution analysis in mRNA level showed that it was highly expressed in gills, hemocytes, and intestine, and lowly expressed in hearts, hepatopancreas, and stomach. Time course expression pattern analysis showed that PcLysM was upregulated in hemocytes and gills after challenge with Vibrio anguillarum, and it was upregulated at 12h after challenge with Staphylococcus aureus in gills. The recombinant PcLysM could bind to different bacteria, and yeast. Further study revealed that PcLysM could bind to peptidoglycans from different bacteria, and chitin. After PcLysM was knocked down, the upregulation of antimicrobial peptide (AMP) genes (crustins and antilipopolysaccharide factors) was suppressed in response to bacterial infection in gills. These results suggest that PcLysM recognizes different microorganisms through binding to polysaccharides, such as peptidoglycans and chitin and regulates the expression of some antimicrobial peptide genes though unknown pathways and regulates the expression of some antimicrobial peptide genes though unknown pathways. This study might provide a clue to elucidate the roles of PcLysM in the innate immune reaction of crayfish P. clarkii.

Catalase eliminates reactive oxygen species and influences the intestinal microbiota of shrimp.

Intestinal innate immune response is an important defense mechanism of animals and humans against external pathogens. The mechanism of microbiota homeostasis in host intestines has been well studied in mammals and Drosophila. The reactive oxygen species (ROS) and antimicrobial peptides have been reported to play important roles in homeostasis. However, how to maintain the microbiota homeostasis in crustacean intestine needs to be elucidated. In this study, we identified a novel catalase (MjCAT) involved in ROS elimination in kuruma shrimp, Marsupenaeus japonicus. MjCAT mRNA was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine. After the shrimp were challenged with pathogenic bacteria via oral infection, the expression level of MjCAT was upregulated, and the enzyme activity was increased in the intestine. ROS level was also increased in the intestine at early time after oral infection and recovered rapidly. When MjCAT was knocked down by RNA interference (RNAi), high ROS level maintained longer time, and the number of bacteria number was declined in the shrimp intestinal lumen than those in the control group, but the survival rate of the MjCAT-RNAi shrimp was declined. Further study demonstrated that the intestinal villi protruded from epithelial lining of the intestinal wall were damaged by the high ROS level in MjCAT-knockdown shrimp. These results suggested that MjCAT participated in the intestinal host-microbe homeostasis by regulating ROS level.