Oral delivery of bacteria expressing wsv108 gene-specific dsRNA protects shrimp from white spot syndrome virus (WSSV) infection.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression by RNA interference and has important application potential in animal disease control. White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp aquaculture, causing huge economic losses every year. In this study, we investigated the function of the WSSV-encoded wsv108 protein. We demonstrated that wsv108 could promote apoptosis by interacting with heat shock protein 70 (HSP70) and enhancing the expression of multiple apoptosis-related genes. Silencing of wsv108 gene by injection with specific dsRNA prepared by in vitro transcription significantly increased the survival rate of WSSV-infected shrimp and reduced the viral load in tissues, suggesting that wsv108 is important for WSSV pathogenicity. Based on this, we expressed the wsv108 specific dsRNA in engineered Escherichia coli. Oral feeding of this bacterium could inhibit the expression of wsv108, increase the survival rate of WSSV-infected shrimp, and decrease the viral load of WSSV in tissues. Therefore, this study developed a new method for treatment of WSSV disease by oral administration of bacterially expressed dsRNA against a novel therapeutic target molecule, which could be a potential candidate strategy for WSSV control in aquaculture.

Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei.

The NF-κB pathway plays an important role in immune regulation. Basigin, an immunoglobulin superfamily membrane protein, is involved in the activation of NF-κB. However, its role in NF-κB signaling in response to pathogen infection remains unclear. In this study, we identified the Basigin gene from Pacific white shrimp, Penaeus vannamei, a representative species for studying the innate immune system of invertebrates. Basigin promoted the degradation of the IκB homolog Cactus, facilitated the nuclear translocation of the NF-κB family member Dorsal, and positively regulated the expression of Dorsal pathway downstream antimicrobial peptide genes. Interestingly, recombinant Basigin protein could bind a variety of Gram-positive and Gram-negative bacteria. Silencing of Basigin inhibited the Dorsal signaling activated by V. parahaemolyticus infection and significantly decreased the survival rate of V. parahaemolyticus-infected shrimp. The expression levels of the antimicrobial peptides ALF1 and ALF2 were downregulated, and the phagocytosis of hemocytes was attenuated in Basigin-silenced shrimp. Similar results were observed in shrimp treated with a recombinant extracellular region of the Basigin protein that was able to compete with endogenous Basigin. Therefore, to the best of our knowledge, this study is the first to demonstrate the function of Basigin as a pathogen recognition receptor that activates NF-κB signaling for antibacterial immunity in shrimp.

The Hippo-Yki pathway downstream transcription factor Scalloped negatively regulates immune defense against Vibrio parahaemolyticus infection in shrimp.

The Hippo-Yki signaling pathway plays a crucial role in numerous biological processes. Previous studies have demonstrated the significance of signal transduction components of the Hippo pathway in the immune response of shrimp. In this study, the downstream transcription factor of Hippo signaling, Scalloped, was analyzed in the context of Vibrio parahaemolyticus infection in Pacific white shrimp, Penaeus vannamei. Upon bacterial and fungal infections, the expression of Scalloped was upregulated in hemocytes. Scalloped was found to localize in the nucleus and interact with the Hippo pathway downstream transcriptional co-activator Yki. With the assistance of Yki, Scalloped activated the promoter of Cactus, a cytoplasmic inhibitor of the NF-κB pathway, leading to the inhibition of the nuclear translocation of the NF-κB family member Dorsal in shrimp. By inhibiting the Dorsal pathway, Scalloped reduced the expression of immune functional proteins and negatively regulated the immune response against bacterial infection in shrimp. RNAi-mediated silencing of Scalloped significantly enhanced the survival rate of V. parahaemolyticus-infected shrimp and reduced the bacterial load in tissues. These findings demonstrate the potential of Scalloped as a therapeutic target for vibriosis in crustaceans and contribute to our understanding of the shrimp's antibacterial defense and the functional roles of Hippo signaling in animal immunity.

White Spot Syndrome Virus (WSSV) Inhibits Hippo Signaling and Activates Yki To Promote Its Infection in Penaeus vannamei.

White spot syndrome virus (WSSV) is a serious threat to shrimp aquaculture, especially Pacific white shrimp, Penaeus vannamei, the most farmed shrimp in the world. Activation of the Hippo-Yki signaling pathway, characterized by the intracellular Hippo-Wts kinase cascade reactions and the phosphorylation and cytoplasmic retention of Yki, is widely involved in various life activities. The current work established the fundamental structure and signal transduction profile of the Hippo-Yki pathway in P. vannamei and further investigated its role in viral infection. We demonstrated that WSSV promoted the dephosphorylation and nuclear translocation of Yki, suggesting that Hippo signaling is impaired and Yki is activated after WSSV infection in shrimp. In vivo, Yki gene silencing suppressed WSSV infection, while Hippo and Wts silencing promoted it, indicating a positive role of Hippo signaling in antiviral response. Further analyses showed that Yki suppressed Dorsal pathway activation and inhibited hemocyte apoptosis in WSSV-infected shrimp, while Hippo and Wts showed opposite effects, which contributed to the role of Hippo signaling in WSSV infection. Therefore, the current study suggests that WSSV annexes Yki to favor its infection in shrimp by inhibiting Hippo signaling. IMPORTANCE White spot syndrome virus (WSSV) is one of the most harmful viral pathogens to shrimp. The pathological mechanism of WSSV infection remains unclear to date. The Hippo-Yki signaling pathway is important for various biological processes and is extensively involved in mammalian immunity, but little is known about its role in infectious diseases in invertebrates. Based on revealing the fundamental structure of the shrimp Hippo pathway, this study investigated its implication in the pathogenesis of WSSV disease. We demonstrated that WSSV enhanced Yki activation by inhibiting Hippo signaling in shrimp. The activated Yki promoted WSSV infection by inhibiting hemocyte apoptosis and suppressing the activation of Dorsal, an NF-κB family member in shrimp that is critical for regulating antiviral response. Therefore, this study suggests that WSSV can hijack the Hippo-Yki signaling pathway to favor its infection in shrimp.

The Hippo-Yki Signaling Pathway Positively Regulates Immune Response against Vibrio Infection in Shrimp.

In the Hippo pathway, activation of Hippo and Warts (Wts) kinases results in the phosphorylation of Yorkie (Yki), to prevent its nuclear translocation. Shrimp aquaculture is threatened by Vibrio genus bacteria. In this study, we examine the role of the Hippo pathway in immune defense against Vibrio parahaemolyticus in Pacific white shrimp Penaeus vannamei. We show that V. parahaemolyticus infection promotes the expression of Yki and facilitates the dephosphorylation and nuclear translocation of Yki, indicating the inhibition of Hippo signaling upon bacterial infection. There is a complex regulatory relationship between the Hippo pathway components Hippo, Wts, and Yki and the immune-related transcription factors Dorsal, Relish, and STAT. Silencing of Hippo and Wts weakened hemocyte phagocytosis, while the silencing of Yki enhanced it, suggesting a positive regulation of shrimp cellular immunity by Hippo signaling activation. In vivo silencing of Hippo and Wts decreased the survival rates of V. parahaemolyticus-infected shrimp and elevated the bacterial content in tissues, while the silencing of Yki showed the opposite results. This suggests that the activation of Hippo signaling and the inhibition of Yki enhance antibacterial immunity in shrimp.

A kelch motif-containing protein KLHDC2 regulates immune responses against Vibrio parahaemolyticus and white spot syndrome virus in Penaeus vannamei.

The kelch motif-containing proteins are widely present in organisms and known to be involved in various biological processes, but their roles in immunity remain unclear. In this study, a kelch motif-containing protein KLHDC2 was identified from Pacific white shrimp Penaeus vannamei and its immune function was investigated. The klhdc2 gene was widely expressed in shrimp tissues and its protein product was mainly present in the nucleus. Expression of klhdc2 was regulated by shrimp NF-κB family members Dorsal and Relish, and changed after immune stimulation. KLHDC2 could enhance the immune defense against Vibrio parahaemolyticus in shrimp but inhibit that against white spot syndrome virus (WSSV). Further analyses showed that KLHDC2 did not affect the phagocytosis of hemocytes but regulated the expression of a series of immune effector genes. KLHDC2 has a complex regulatory relationship with Dorsal and Relish, which may partly contribute to its positive role in antibacterial response by regulating humoral immunity. Moreover, the regulatory effect of KLHDC2 on WSSV ie1 gene contributed to its negative effect on antiviral response. Therefore, the current study enrichs the knowledge on the Kelch family and helps to learn more about the regulatory mechanism of shrimp immunity.

Wnt5b plays a negative role in antibacterial response in Pacific white shrimp Penaeus vannamei.

The Wnt family genes are essentially implicated in development and growth in animals. Accumulating clues have pointed to the importance of Wnts in invertebrate immunity, but the underlying mechanisms are still unclear to date. The Wnt5b has been known to promote white spot syndrome virus (WSSV) infection in shrimp but its role in antibacterial response remains unclear. In the current study, we focused on the involvement of Wnt5b in Vibrio parahaemolyticus infection in Pacific white shrimp Penaeus vannamei. We demonstrated that the expression of Wnt5b was regulated by the IMD-Relish and JAK-STAT pathways but not the Dorsal pathway and was suppressed upon bacterial infection. Although Wnt5b did not affect the cellular immunity in shrimp, it was involved in regulation of humoral immunity. Silencing of Wnt5b in vivo significantly increased expression of several antimicrobial peptides but decreased that of many immune functional proteins including C-type lectins and lysozymes. Treatment with recombinant Wnt5b protein increased the susceptibility of shrimp to V. parahaemolyticus infection, while silencing of Wnt5b in vivo showed an opposite result. These suggested that Wnt5b plays a negative role in antibacterial response in shrimp. Together with previous reports, the current study shows that Wnt5b functions as an inhibitor for shrimp immunity, which is a potential target for improving immune responses against infection.

Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell.

Background: High mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption. Methods: In the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms. Results: In the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs. Conclusion: HMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.

The LARK protein is involved in antiviral and antibacterial responses in shrimp by regulating humoral immunity.

The LARK proteins containing a C2HC-type zinc finger motif and two RNA recognition motifs are conserved across vertebrates and invertebrates. Previous studies have suggested that invertebrate LARKs and their mammalian counterparts, the RBM4 proteins, regulate gene expression by affecting RNA stability and post-transcriptional processing, participating in multiple life processes. In the current study, the LARK gene from Pacific white shrimp Litopenaeus vannamei was identified and functionally explored in the context of immunity. The LARK protein was mainly present in the nucleus of its expression vector-transfected S2 cells, and the LARK mRNA was detectable in all the tested shrimp tissues. Expression of LARK in gill was up-regulated by immune stimulation with various pathogens. In vivo experiments demonstrated that LARK played positive roles in both antiviral and antibacterial responses and silencing of LARK could make shrimp more susceptible to infection with Vibrio parahaemolyticus and white spot syndrome virus (WSSV). Although silencing of LARK did not affect the phagocytic activity of hemocytes, it regulated expression of many components of the NF-κB and JAK-STAT pathways and a series of immune function proteins. These suggested that LARK could be mainly involved in regulation of humoral immunity. The current study could help reveal the roles of LARK/RBM4 in immunity and further explore the regulatory mechanisms of shrimp immunity.

A novel C-type lectin with microbiostatic and immune regulatory functions from Litopenaeus vannamei.

C-type lectins (CTLs) are a group of lectins with at least one carbohydrate recognition domain (CRD), the binding of which to carbohydrates requires the presence of calcium ions. CTLs generally function as pattern recognition receptors (PRRs), essentially participating in innate immunity. In the current study, a novel CTL termed LvCTL5 was identified from Pacific white shrimp Litopenaeus vannamei, which shared sequence identities with other crustacean CTLs. LvCTL5 was highly expressed in hepatopancreas and could be activated by infection with bacteria, virus and fungi. The recombinant LvCTL5 protein purified from E. coli showed microbiostatic and agglutination activities against bacteria and fungi in vitro. Silencing of LvCTL5 in vivo could significantly affect expression of a series of immune effector genes and down-regulate the phagocytic activity of hemocytes. Compared with controls, the LvCTL5-silenced shrimp were highly susceptible to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infections. These suggest that LvCTL5 has microbiostatic and immune regulatory activities and is implicated in antiviral and antibacterial responses.