Subcellular Translocation of Yorkie through the PRP4K-CRM1 Axis Regulates Antimicrobial Peptides Transcription and Defense against Bacterial Infection in Crab.

The Hippo signaling pathway plays important roles in innate immunity. In the current study, we found that bacterial infection did not influence mRNA and protein levels of yorkie (Yki), which is an important terminal molecule of the Hippo signaling pathway. However, bacterial infection promoted the translocation of Yki from the nucleus to the cytoplasm in Chinese mitten crab (Eriocheir sinensis), thus attenuating Yki-suppressed transcription of antimicrobial peptides through Cactus. Chromosome region maintenance 1 (CRM1)-silenced crab hemocytes significantly suppressed Yki translocation from the nucleus to the cytoplasm upon bacterial infection, resulting in significantly increased expression of Cactus, decreased expression of antimicrobial peptides, and higher bacterial susceptibility, which demonstrated the regulatory role of CRM1 in subcellular localization of Yki. However, RNA interference of Scalloped (Sd) exhibited no effect on the subcellular localization of Yki and its regulation of Cactus/antimicrobial peptides. Moreover, we elucidated that both CRM1 and Sd could interact with Yki and that the PRP4K-mediated phosphorylation of a conserved serine amino acid residue in the nuclear export signal of Yki is essential for interaction between Yki and CRM1; however, the phosphorylation did not affect the binding of Yki with Sd. We also found that bacterial infection significantly promoted the expression of PRP4K in hemocytes; RNA interference of PRP4K and phosphatase inhibitor suppressed Yki translocation from the nucleus to the cytoplasm, promoting Cactus expression and inhibiting antimicrobial peptide expression. Thus, subcellular localization of Yki regulates antibacterial infection through both PRP4K and CRM1 in crabs.

Caspar negatively regulates anti-bacterial immunity by controlling the nuclear translocation of Relish in Chinese mitten crab (Eriocheir sinensis).

Caspar, a homolog of the Fas-associated factor 1 (FAF1) family, contains an N-terminal ubiquitin interaction domain, a ubiquitin-like self-association domain, and a C-terminal ubiquitin regulatory domain. Caspar has been reported to be involved in the antibacterial immunity of Drosophila, which is unclear whether it is involved in the antibacterial immune process of crustaceans. In this article, we identified a Caspar gene in Eriocheir sinensis and named it EsCaspar. EsCaspar positively respond to bacterial stimulation and downregulate the expression of certain associated antimicrobial peptides by inhibiting the nuclear translocation of EsRelish. Thus, EsCaspar might be a suppressor of the immune deficiency (IMD) pathway that prevents over-activation of the immune system. Indeed, excess EsCaspar protein in crabs reduced resistance to bacterial infection. In conclusion, EsCaspar is a suppressor of the IMD pathway in crabs that plays a negative regulatory role in antimicrobial immunity.

A new SVWC protein functions as a pattern recognition protein in antibacterial responses in Chinese mitten crab (Eriocheirsinensis).

Because invertebrates lack acquired immunity, they rely primarily on the innate immune system to defend themselves against viral and bacterial infections. SVWC, also called Vago, is a class of small-molecule proteins characterized by a single von Willebrand factor C-domain and appears to be restricted to arthropods. It has been reported that SVWC is involved in antiviral immunity in invertebrates, but whether it is involved in antimicrobial immunity and the mechanism of its involvement in antimicrobial immunity remains unclear. In this study, we identified a novel SVWC gene in Eriocheir sinensis and named it EsSVWC. EsSVWC was found to respond positively to bacterial stimulation and to regulate the expression of related antimicrobial peptides (AMPs). The EsSVWC protein recognized and bound to a variety of pathogen-associated molecular patterns (PAMPs) but did not exhibit direct bactericidal effects. Thus, the EsSVWC protein in crabs helps resist bacterial infection and improve survival rates. In summary, EsSVWC may regulate the innate immune system of crabs in response to microbial invasion in an indirect manner.

Antibacterial responses and functional characterization of the interferon gamma inducible lysosomal thiol reductase (GILT) protein in Chinese mitten crab (Eriocheir sinensis).

The inducible reductase of interferon gamma (IFN- γ), IFN-γ-induced lysosomal thiol reductase (GILT) is important in antiviral immunity, but its mechanism in invertebrate antimicrobial immunity is unclear. We determined that GILT protein was involved in the antibacterial immunity of Chinese mitten crab (Eriocheir sinensis). GILT protein was highly expressed in crab hemocytes and was significantly upregulated 6 h after bacterial stimulation. Recombinant E. sinensis GILT (rEsGILT) contained a CXXS active site that catalyzed disulfide bond reduction. Vibrio parahaemolyticus and Staphylococcus aureus were bound through interaction with peptidoglycan and lipopolysaccharide, respectively, and bacterial agglutination and clearance in the crabs was markedly promoted. Nevertheless, EsGILT exhibited no direct antibacterial or bactericidal activity. EsGILT also promoted crab hemocyte phagocytosis and played an anti-bacterial role, and inhibited hemocyte apoptosis. In summary, EsGILT promoted bacterial agglutination, clearance, and phagocytosis by recognizing and agglutinating pathogenic microorganisms and reduced the apoptosis level, indirectly participating in antibacterial reactions.

The role of ficolin as a pattern recognition receptor in antibacterial immunity in Eriocheir sinensis.

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named EsFicolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. EsFicolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant EsFicolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant EsFicolin could effectively remove bacteria and showed direct antibacterial activity. EsFicolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that EsFicolin plays an important role in the crab antibacterial immune response.

Effect of polystyrene nanoplastics on cell apoptosis, glucose metabolism, and antibacterial immunity of Eriocheir sinensis.

The adverse effects of plastic waste and nanoplastics on the water environment have become a focus of global attention in recent years. In the present study, using adult Chinese mitten crabs (Eriocheir sinensis) as an animal model, the bioaccumulation and the in vivo and in vitro toxicity of polystyrene nanoplastics (PS NPs), alone or in combination with the bacteria, were investigated. This study aimed to investigate the effects of PS NPs on apoptosis and glucose metabolism in Chinese mitten crabs, and whether PS NPs could synergistically affect the antibacterial immunity of crabs. We observed that NPs were endocytosed by hemocytes, which are immune cells in crustaceans and are involved in innate immunity. The RNA sequencing data showed that after hemocytes endocytosed NPs, apoptosis and glucose metabolism-related gene expression was significantly induced, resulting in abnormal cell apoptosis and a glucose metabolism disorder. In addition, exposure to NPs resulted in changes in the antimicrobial immunity of crabs, including changes in antimicrobial peptide expression, survival, and bacterial clearance. In summary, NPs could be endocytosed by crab hemocytes, which adversely affected the cell apoptosis, glucose metabolism, and antibacterial immunity of Eriocheir sinensis. This study revealed the effects of NPs on crab immunity and lays the foundation for further exploration of the synergistic effect of NPs and bacteria.

A Novel Ig Domain-Containing C-Type Lectin Triggers the Intestine-Hemocyte Axis to Regulate Antibacterial Immunity in Crab.

The C-type lectin family with the signature C-type lectin-like domain promotes antibacterial host defense within the animal kingdom. We examined the role of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards) (Decapoda: Grapsidae) Ig domain-containing C-type lectin (EsIgLectin), a novel and poorly understood member of the C-type lectin family. EsIgLectin was expressed primarily by both hemocytes (E sinensis) and intestines, with significantly induced mRNA expression on intestinal or hemolymph bacterial infections. As a soluble protein, both its C-type lectin-like domain and the Ig domain were required for bacterial binding, bacterial agglutination, bacterial growth inhibition, and in vivo bacterial clearance. Polymeric EsIgLectin could be constructed via the disulfide bond in the Ig domain, significantly enhancing EsIgLectin antibacterial activity. EsIgLectin promoted bacterial phagocytosis in an Ig domain-dependent manner in hemocytes, while it controlled microbial homeostasis and protected against bacteria-induced inflammation in the intestine. Protein interaction studies revealed that the EsIgLectin Ig domain bound to the first Ig domain of the polymeric Ig receptor, which was essential for EsIgLectin-induced bacterial phagocytosis. The temporal sequence of cell interactions during intestinal inflammation is only beginning to be understood. In this article, we show that hemocyte-derived EsIgLectin entered the intestinal wall at the later phase of intestinal inflammation. Moreover, EsIgLectin protected the host against intestinal and hemolymph infections in a polymeric Ig receptor-dependent manner. Therefore, the EsIgLectin promoted bacterial clearance and protected against inflammatory disease through an independent or synergistic effect of hemocytes and intestines in invertebrates.

A novel ML protein functions as a pattern recognition protein in antibacterial responses in Eriocheir sinensis.

The myeloid differentiation factor 2 (MD-2)-related lipid recognition (ML) domain is present in MD-2, MD-1, GM2-activator protein (GM2A) and Niemann-Pick disease type C2 (NPC2). ML proteins function in antibacterial signal transduction and lipid metabolism in vertebrates, but the mechanism in invertebrates is unknown. In this study, we found that ML proteins were involved in bacterial resistance in Chinese mitten crab (Eriocheir sinensis). One member, EsML3, a soluble, bacterial-induced pattern recognition protein was upregulated in hemocytes following bacterial challenge. Recombinant EsML3 bound to Gram-negative bacteria (Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) by interaction with peptidoglycan, lipopolysaccharide. EsML3 showed no direct bacteriostatic or bacteriocidal activity. Pre-incubating bacteria with rEsML3 significantly promoted in vivo bacterial clearance. EsML3 also promoted phagocytic activity and plays a role against bacterial infection. In summary, EsML3 mediates cellular immune responses by recognising invasive microorganisms, promoting bacterial clearance and phagocytosis against bacterial infection in crab.

Distinct vitellogenin domains differentially regulate immunological outcomes in invertebrates.

The classical role of Vitellogenin (Vg) is providing energy reserves for developing embryos, but its roles appear to extend beyond this nutritional function, and its importance in host immune defense is garnering increasing research attention. However, Vg-regulated immunological functions are dependent on three different domains within different species and remain poorly understood. In the present study, we confirmed three conserved VG domains-LPD_N, DUF1943, and VWD-in the Chinese mitten crab (Eriocheir sinensis), highlighting functional similarities of Vg in vertebrates and invertebrates. Of these three domains, DUF1943 and VWD showed definitive bacterial binding activity via interaction with the signature components on microbial surfaces, but this activity was not exhibited by the LPD_N domain. Antibacterial assays indicated that only the VWD domain inhibits bacterial proliferation, and this function may be conserved between different species due to the conserved amino acid residues. To further explore the relationship between Vg and polymeric immunoglobulin receptor (pIgR), we expressed EspIgR and the three E. sinensis Vg (EsVg) domains in HEK293T cells, and coimmunoprecipitation assay demonstrated that only the DUF1943 domain interacts with EspIgR. Subsequent experiments demonstrated that EsVg regulates hemocyte phagocytosis by binding with EspIgR through the DUF1943 domain, thus promoting bacterial clearance and protecting the host from bacterial infection. To the best of our knowledge, our work is the first to report distinct domains in Vg inducing different immunological outcomes in invertebrates, providing new evidence that pIgR acts as a phagocytic receptor for Vg.

Bacteria-induced IMD-Relish-AMPs pathway activation in Chinese mitten crab.

The innate immune response is an important line of defense against invading pathogens in invertebrates. Signaling pathways, including the IMD pathway, play critical roles in the production of antimicrobial peptides (AMPs), which induce the transcription of immune effectors that protect against bacterial invasion. In the present study, the cDNA of IMD from Eriocheir sinensis was cloned (designated EsIMD) and shown to be significantly upregulated following Gram-positive and Gram-negative bacterial infection. In vivo and in vitro studies collectively suggested that both the Gram-negative bacterium Vibrio parahemolyticus and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis elicit the translocation of Relish. Moreover, EsIMD positively regulated EsRelish translocation from the cytoplasm to the nucleus following stimulation with both Gram-positive and Gram-negative bacteria. EsRelish knockdown in hemocytes significantly suppressed AMPs' expression. Furthermore, both Lys-type and DAP-type peptidoglycan-containing bacteria activated the IMD pathway and elicited antibacterial responses in crab. Conclusively, these findings demonstrate that both Gram-positive and Gram-negative bacteria activate IMD signaling, via a mechanism that is distinct with that by which Gram-negative bacteria activate IMD signaling in Drosophila. These findings might pave the way for a better understanding of the innate immune system and the fundamental network of the IMD signaling pathway in crustacean.

C-type lectin response to bacterial infection and ammonia nitrogen stress in tiger shrimp (Penaeus monodon).

C-type lectins (CTLs) are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many shrimp, C-type lectins subfamily contain a large number of members with different functions that need to research in deep. In this present study, a new type of CTL, PmCL1 with 861 bp long full-length cDNA, that encodes a protein with 164-amino acid from a 495-bp open reading frame, was isolated and characterized from tiger shrimp (Penaeus monodon). The mRNA transcript of PmCL1 showed the highest expression in the hepatopancreas, whereas it was barely detected in the ovary. After the shrimp were stimulated by Vibrio harveyi and Vibrio anguillarum, PmCL1 expression in the hepatopancreas and gill was significantly upregulated. A carbohydrate-binding assay revealed the specificity of PmCL1 for pathogen-associated molecular patterns (PAMPs) that included peptidoglycan (PGN) and lipopolysaccharide (LPS), and saccharides that included d-glucose, galactosamine, α-lactose, treholose, and d-mannose. Recombinant PmCL1 agglutinated gram-positive (Staphylococcus aureus) and gram-negative bacteria (V. harveyi, V. anguillarum, Vibrio alginolyticus, Vibrio parahemolyticus, Vibrio vulnificus, and Aeromonas hydrophila) in the presence of calcium ions and enhanced the efficiency of clearing the invading bacteria. Collectively, our results suggested that PmCL1 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, as well as the response towards ammonia nitrogen stress.