Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans.

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Metabolomic Profiles in the Intestine of Shrimp Infected by White Spot Syndrome Virus and Antiviral Function of the Metabolite Linoleic Acid in Shrimp.

White spot syndrome virus (WSSV) is a threatening pathogenic virus in shrimp culture, and at present, no effective strategy can prevent and control the disease. Intestinal flora and its metabolites are important for the resistance of shrimp to lethal pathogenic viruses. However, the changes of metabolites in the shrimp intestines after WSSV infection remain unclear. We established an artificial oral infection method to infect shrimp with WSSV and analyzed the metabolites in intestinal content of shrimp by HPLC and tandem mass spectrometry. A total of 78 different metabolites and five different metabolic pathways were identified. Among them, we found that the content of linoleic acid, an unsaturated fatty acid, increased significantly after WSSV infection, indicating that linoleic acid might be involved in antiviral immunity in shrimp. Further study showed that, after oral administration of linoleic acid, WSSV proliferation decreased evidently in the shrimp, and survival rate of the shrimp increased significantly. Mechanical analysis showed that linoleic acid directly bound to WSSV virions and inhibited the viral replication. Linoleic acid also promoted the expression of antimicrobial peptides and IFN-like gene Vago5 by activating the ERK-NF-κB signaling pathway. Our results indicated that WSSV infection caused metabolomic transformation of intestinal microbiota and that the metabolite linoleic acid participated in the immune response against WSSV in shrimp.

FOXO regulates the expression of antimicrobial peptides and promotes phagocytosis of hemocytes in shrimp antibacterial immunity.

Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp (Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly or indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates.

Mitochondrial ATPase inhibitor factor 1, MjATPIF1, is beneficial for WSSV replication in kuruma shrimp (Marsupenaeus japonicus).

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.

The polymeric immunoglobulin receptor-like protein from Marsupenaeus japonicus is a receptor for white spot syndrome virus infection.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

Thymosins participate in antibacterial immunity of kuruma shrimp, Marsupenaeus japonicus.

Thymosins ß are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp.

Protein Inhibitor of Activated STAT (PIAS) Negatively Regulates the JAK/STAT Pathway by Inhibiting STAT Phosphorylation and Translocation.

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

Scavenger receptor C promotes bacterial clearance in kuruma shrimp Marsupenaeus japonicus by enhancing hemocyte phagocytosis and AMP expression.

Scavenger receptors (SRs) comprise a large family of structurally diverse glycoproteins located on the cell membrane and function as pattern-recognition receptors (PRRs) participating in innate immunity in different species. Class C scavenger receptor (SRC) has been only identified in invertebrates and its biological functions still need to be researched. In this study, we characterized the anti-bacterial function of a SRC from kuruma shrimp Marsupenaeus japonicus (MjSRC). The mRNA level of MjSRC was up-regulated significantly in hemocytes of kuruma shrimp challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant extracellular domains (MAM and CCP domains) of MjSRC have the ability of binding different bacteria and glycans in vitro. After knockdown of MjSRC, the bacterial clearance ability and phagocytic rate of hemocyte decreased significantly in vivo. Meanwhile, overexpression of MjSRC in shrimp enhanced the clearance ability and phagocytic rate of hemocytes. Further study found that MjSRC could regulate the expression of several antimicrobial peptides (AMPs). All these results indicate that MjSRC plays important roles in antibacterial immunity in kuruma shrimp by enhancing hemocyte phagocytosis and AMP expression.

Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp.

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjß-arrestin2. Further studies found that Mjß-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus.

Involvement of a LysM and putative peptidoglycan-binding domain-containing protein in the antibacterial immune response of kuruma shrimp Marsupenaeus japonicus.

Lysin motif (LysM) is a peptidoglycan and chitin-binding motif with multiple functions in bacteria, plants, and animals. In this study, a novel LysM and putative peptidoglycan-binding domain-containing protein was cloned from kuruma shrimp (Marsupenaeus japonicus) and named as MjLPBP. The cDNA of MjLPBP contained 1010 nucleotides with an open reading frame of 834 nucleotides encoding a protein of 277 amino acid residues. The deduced protein contained a Lysin motif and a transmembrane region, with a calculated molecular mass of 31.54 kDa and isoelectric point of 8.61. MjLPBP was ubiquitously distributed in different tissues of shrimp at the mRNA level. Time course expression assay showed that MjLPBP was upregulated in hemocytes of shrimp challenged with Vibrio anguillarum or Staphylococcus aureus. MjLPBP was also upregulated in hepatopancreas after white spot syndrome virus and bacteria challenge. The recombinant protein of MjLPBP could bind to some Gram-positive and Gram-negative bacteria and yeast. Further study found that rMjLPBP bound to bacterial cell wall components, including peptidoglycans, lipoteichoic acid, lipopolysaccharide, and chitin. The induction of several antimicrobial peptide genes and phagocytosis-related gene, such as anti-lipopolysaccharide factors and myosin, was depressed after knockdown of MjLPBP. MjLPBP could facilitate V. anguillarum clearance in vivo. All the results indicated that MjLPBP might play an important role in the innate immunity of shrimp.

Dual oxidases participate in the regulation of intestinal microbiotic homeostasis in the kuruma shrimp Marsupenaeus japonicus.

The metazoan gut lumen harbors numerous microbial communities. Tolerance for high bacterial counts and maintenance of microbiota homeostasis remain insufficiently studied. In this study, we identified a novel dual oxidase (MjDUOX2) involved in reactive oxygen species (ROS) production in the kuruma shrimp Marsupenaeus japonicus. MjDUOX2 is a transmembrane protein with an N-signal peptide region (19 aa) and a peroxidase homology domain (PHD, 554 aa) in the extracellular region; seven transmembrane regions; and three EF (calcium-binding region) domains (110 aa), a FAD-binding domain (104 aa), and a NAD-binding domain (156 aa) in the intracellular region. The novel MjDUOX2 exhibits a relatively low similarity (26.84% identity) to a previously reported DUOX in the shrimp (designated as MjDUOX1). The mRNA of MjDUOXs was widely distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Oral infection of the shrimp with pathogenic bacteria upregulated the mRNA expression of MjDUOXs and increased the ROS level in the intestine. However, High ROS level could inhibit the expression of MjDUOXs in shrimp after Vibrio anguillarum infection. Knockdown of MjDUOXs by RNA interference (RNAi) decreased the ROS level, increased the bacterial count in the intestine, and decreased the survival rate of the MjDUOX-RNAi shrimp infected with V. anguillarum. These results suggest that MjDUOXs play an important role for microbiota homeostasis in intestine of shrimp.

ß-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 ofDrosophila melanogasterand 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 againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that ß-arrestins negatively regulate Toll signaling in two different ways. ß-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 ß-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. ß-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 Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that ß-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

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.

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.