Cytokine-like-Vago-mediated antiviral response in Penaeus monodon via IKK-NF-κB signaling pathway.

Interferon (IFN) system is the primary mechanism of innate antiviral defense in immune response. To date, limited studies of IFN system were conducted in crustaceans. Previous report in Penaeus monodon demonstrated the interconnection of cytokine-like molecule Vago and inhibitor of kappa B kinase-nuclear factor κB (IKK-NF-κB) cascade against white spot syndrome virus (WSSV). This study further identified five different PmVago isoforms. Upon immune stimulation, PmVagos expressed against shrimp pathogens. PmVago1, PmVago4, and PmVago5 highly responded to WSSV, whereas, PmVago1 and PmVago4 RNAi exhibited a rapid mortality with elevated WSSV replication. Suppression of PmVago1 and PmVago4 negatively affected proPO system, genes in signal transduction, and AMPs. WSSV infection additionally induced PmVaog4 granule accumulation and cellular translocation to the area of cell membrane. More importantly, PmVago1 and PmVago4 promoters were stimulated by PmIKK overexpression; meanwhile, they further activated Dorsal and Relish promoter activities. These results suggested the possible roles of the cytokine-like PmVago via IKK-NF-κB cascade against WSSV infection.

Molecular characterization of turtle-like protein in whiteleg shrimp (Litopenaeus vannamei) and its role in Enterocytozoon hepatopenaei infection.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects shrimp hepatopancreas, causing growth retardation and disease susceptibility. Knowledge of the host-pathogen molecular mechanisms is essential to understanding the microsporidian pathogenesis. Turtle-like protein (TLP) is part of the immunoglobulin superfamily of proteins, which is widely distributed in the animal kingdom. TLP has multiple functions, such as cell surface receptors and cell adhesion molecules. The spore wall proteins (SWPs) of microsporidia are involved in the infection mechanisms. Some SWPs are responsible for spore adherence, which is part of the activation and host cell invasion processes. Previous studies showed that TLP from silkworms (Bombyx mori) interacted with SWP26, contributing to the infectivity of Nosema bombycis to its host. In this study, we identified and characterized for the first time, the Litopenaeus vannamei TLP gene (LvTLP), which encodes an 827-aa protein (92.4 kDa) composed of five immunoglobulin domains, two fibronectin type III domains, and a transmembrane region. The LvTLP transcript was expressed in all tested tissues and upregulated in the hepatopancreas at 1 and 7 days post-cohabitation (dpc) and at 9 dpc in hemocytes. To identify the LvTLP binding counterpart, recombinant (r)LvTLP and recombinant (r)EhSWP1 were produced in Escherichia coli. Coimmunoprecipitation and enzyme-linked immunosorbent assays demonstrated that rLvTLP interacted with rEhSWP with high affinity (KD = 1.20 × 10-7 M). In EHP-infected hepatopancreases, LvTLP was clustered and co-localized with some of the developing EHP plasmodia. Furthermore, LvTLP gene silencing reduced the EHP copy numbers compared with those of the control group, suggesting the critical role of LvTLP in EHP infection. These results provide insight into the molecular mechanisms of the host-pathogen interactions during EHP infection.

Role of hemocytin from Litopenaeus vannamei in immune response against microsporidian, Enterocytozoon hepatopenaei.

Hemocytin, a multidomain hemostasis-related protein, is a homologous protein of hemolectin in Drosophila melanogaster and von Willebrand factor (vWF) in humans. The vWF type D (VWD) domain in hemocytin is thought to be a major mediator of hemocyte aggregation and the prophenoloxidase (proPO) activation system. Here, we report for the first time the role of hemocytin from Litopenaeus vannamei (LvHCT) against Enterocytozoon hepatopenaei (EHP), the pathogenic microsporidian causing hepatopancreatic microsporidiosis in Pacific white shrimp (L. vannamei). The LvHCT gene contains 58,366 base pairs consisting of 84 exons encoding for 4267 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that LvHCT was clustered with crustacean hemocytins. Gene expression analysis by quantitative real-time RT-PCR showed that LvHCT in hemocytes was significantly upregulated at 9 and 11 days post-EHP cohabitation, which was consistent with EHP copy numbers in the infected shrimp. To further investigate the biological function of LvHCT in EHP infection, a recombinant protein containing an LvHCT-specific VWD domain (rLvVWD) was expressed in Escherichia coli. In vitro agglutination assays showed that rLvVWD was functionally representative of LvHCT and induced aggregation of pathogens, including Gram-negative and -positive bacteria, fungi, and EHP spore. LvHCT suppression resulted in higher EHP copy numbers and proliferation due to the lack of hemocytin-mediated EHP spore aggregation in LvHCT-silenced shrimp. Moreover, immune-related genes in the proPO-activating cascade and Toll, IMD and JAK/STAT signaling pathways were upregulated to eliminate the over-controlled EHP in LvHCT-silenced shrimp. Furthermore, the impaired phenoloxidase activity due to LvLGBP suppression was recovered after rLvVWD injection, suggesting that LvHCT may be directly involved in phenoloxidase activation. In conclusion, a novel LvHCT is involved in shrimp immunity against EHP via EHP spore aggregation and possible activation of the proPO-activating cascade.