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.

Heat shock protein 90 of Pacific white shrimp (Litopenaeus vannamei) is possibly involved in promoting white spot syndrome virus infection.

Viruses cause up to 60% of disease-associated losses in shrimp aquaculture, and the white spot syndrome virus (WSSV) is a major viral pathogen in shrimp. Heat shock proteins (HSPs) are host chaperones that help promote many viral infections. We investigated the involvement of Litopenaeus vannamei (Lv) HSP90 in WSSV infections. Expression of LvHSP90 at the transcript and protein levels were upregulated after WSSV infection. Silencing LvHSP90 resulted in the increased cumulative mortality rate and the reduction of circulating hemocytes. The inhibition of LvHSP90 also induced the expression of apoptosis-related genes which indicated the induction of apoptotic pathway and might lead to shrimp death. However, lower the number of WSSV-infected cells and viral copy numbers were detected in the LvHSP90-silenced shrimp compared with those of the controls, corresponding with significantly decreased expressions of viral genes, including the immediate-early genes WSV083 and WSV249 and viral DNA polymerase. Conversely, injecting shrimp with WSSV that had been co-incubated with a recombinant LvHSP90 (rLvHSP90) promoted WSSV infection as evidenced by an increased cumulative mortality rate and viral copy numbers at 40-48 h post infection (hpi). Subcellular localization of LvHSP90 in WSSV-infected hemocytes at 3, 6 and 12 hpi demonstrated increased expression and translocation of LvHSP90 into the nucleus where WSSV DNA can replicate. Thus, LvHSP90 might be involved in the WSSV pathogenesis by promoting WSSV replication.

Heat Shock Protein 70 Is a Damage-Associated Molecular Pattern That by Binding to Lipopolysaccharide and ß-1,3-Glucan-Binding Protein Activates the Prophenoloxidase System in Shrimp.

Recent studies have initiated a paradigm shift in understanding heat shock protein 70 (HSP70) functions in the shrimp immune system. However, the mechanism by which Litopenaeus vannamei (Lv)HSP70 modulates the innate immune response remains unclear. This study shows that LvHSP70 binds to the pattern recognition receptor LPS and ß-1,3-glucan-binding protein (LvLGBP), and subsequently leads to the activation of the prophenoloxidase system. Injection of shrimp with rLvHSP70 significantly (p < 0.05) upregulated the gene and protein expression of the key pattern recognition receptor LvLGBP. A coimmunoprecipitation and ELISA-based binding assay strongly confirmed the binding of LvHSP70 to LvLGBP at polysaccharide recognition motifs (PLS motifs) with a Kd of 4.44 µM and its competitive binding with LPS (IC50) is 8.036 µM. Conversely, LPS efficiently competed with LvHSP70 for binding to LvLGBP in a concentration-dependent manner with an IC50 of 7.662 µM, indicating that both are ligands of LvLGBP and likely bind at the same site. Binding of LvHSP70 to LvLGBP highly activated phenoloxidase activity in shrimp hemocyte lysate supernatants. Gene silencing of LvLGBP impaired the activation of phenoloxidase activity in shrimp by rLvHSP70, indicating that LvHSP70-LvLGBP interaction was essential for stimulating the immune cascade. Taken together, these results demonstrated that LvHSP70 is a ligand of LvLGBP similar to LPS and acts as a damage-associated molecular pattern to modulate the shrimp immune system via the prophenoloxidase system, eventually leading to the production of melanin and toxic reactive intermediates against invading pathogens.

Structure, gene expression, and putative functions of crustacean heat shock proteins in innate immunity.

Heat shock proteins (HSPs) are molecular chaperones with critical roles in the maintenance of cellular proteostasis. HSPs, which regulate protein folding and refolding, assembly, translocation, and degradation, are induced in response to physiological and environmental stressors. In recent years, HSPs have been recognized for their potential role in immunity; in particular, these proteins elicit a variety of immune responses to infection and modulate inflammation. This review focuses on delineating the structural and functional roles of crustacean HSPs in the innate immune response. Members of crustacean HSPs include high molecular weight HSPs (HSP90, HSP70, and HSP60) and small molecular weight HSPs (HSP21 and HSP10). The sequences and structures of these HSPs are highly conserved across various crustacean species, indicating strong evolutionary links among this group of organisms. The expression of HSP-encoding genes across different crustacean species is significantly upregulated upon exposure to a wide range of pathogens, emphasizing the important role of HSPs in the immune response. Functional studies of crustacean HSPs, particularly HSP70s, have demonstrated their involvement in the activation of several immune pathways, including those mediating anti-bacterial resistance and combating viral infections, upon heat exposure. The immunomodulatory role of HSPs indicates their potential use as an immunostimulant to enhance shrimp health for control of disease in aquaculture.

Sequence and expression analysis of HSP70 family genes in Artemia franciscana.

Thus far, only one gene from the heat shock protein 70 (HSP70) family has been identified in Artemia franciscana. Here, we used the draft Artemia transcriptome database to search for other genes in the HSP70 family. Four novel HSP70 genes were identified and designated heat shock cognate 70 (HSC70), heat shock 70 kDa cognate 5 (HSC70-5), Immunoglobulin heavy-chain binding protein (BIP), and hypoxia up-regulated protein 1 (HYOU1). For each of these genes, we obtained nucleotide and deduced amino acid sequences, and reconstructed a phylogenetic tree. Expression analysis revealed that in the juvenile state, the transcription of HSP70 and HSC70 was significantly (P < 0.05) higher in a population of A. franciscana selectively bred for increased induced thermotolerance (TF12) relative to a control population (CF12). Following non-lethal heat shock treatment at the nauplius stage, transcription of HSP70, HSC70, and HSC70-5 were significantly (P < 0.05) up-regulated in TF12. In contrast, transcription of the other HSP70 family members in A. franciscana (BIP, HYOU1, and HSPA4) showed no significant (P > 0.05) induction. Gene expression analysis demonstrated that not all members of the HSP70 family are involved in the response to heat stress and selection and that especially altered expression of HSC70 plays a role in a population selected for increased thermotolerance.

Litopenaeus vannamei heat shock protein 70 (LvHSP70) enhances resistance to a strain of Vibrio parahaemolyticus, which can cause acute hepatopancreatic necrosis disease (AHPND), by activating shrimp immunity.

Heat shock protein 70 (HSP70) acts as a molecular chaperone and a stress protein, but also plays important roles in innate and adaptive immune responses. Previous studies have reported that non-lethal heat shock (NLHS) could enhance the resistance of Pacific white shrimp Litopenaeus vannamei to a specific strain of Vibrio parahaemolyticus, which carried a toxin-producing plasmid (VPAHPND), via the induction of LvHSP70 transcription. Here, we further investigated the specific function of LvHSP70 in shrimp immunity. The upregulation of LvHSP70 at the protein level was detected during recovery time after NLHS treatment, using both western blot analysis and immunofluorescence microscopy. We found that NLHS immediately activated the production of LvHSP70 in shrimp hemocytes and that such induction was observed in all three types of hemocytes: hyaline; granular and semi-granular cells. Furthermore, the role of LvHSP70 in bacterial defense was investigated using the heterologous expression of recombinant LvHSP70 (rLvHSP70) in Escherichia coli. Shrimp receiving rLvHSP70 by injection showed an increased survival rate (75%) to VPAHPND infection compared to just 20% survival in the control group injected with bovine serum albumin (BSA). We also demonstrated that the injected rLvHSP70 accumulated in shrimp hemocytes and was detected in the intracellular space of hemocyte cells leading to the induced expression (P<0.05) of several immune-related genes (LvMyD88, LvIKKß, LvIKKε, LvCrustin I, LvPEN2, LvPEN3, LvproPO1, LvproPO2 and LvTG1). Collectively, these results suggest that LvHSP70 plays a crucial role in bacterial defense by activating the shrimp immune system.

Heat shock factor 1 regulates heat shock proteins and immune-related genes in Penaeus monodon under thermal stress.

Heat shock factors (HSFs) participate in the response to environmental stressors and regulate heat shock protein (Hsp) expression. This study describes the molecular characterization and expression of PmHSF1 in black tiger shrimp Penaeus monodon under heat stress. PmHSF1 expression was detected in several shrimp tissues: the highest in the lymphoid organ and the lowest in the eyestalk. Significant up-regulation of PmHSF1 expression was observed in hemocytes (p < 0.05) following thermal stress. The expression of several PmHsps was rapidly induced following heat stress. Endogenous PmHSF1 protein was expressed in all three types of shrimp hemocyte and strongly induced under heat stress. The suppression of PmHSF1 expression by dsRNA-mediated gene silencing altered the expression of PmHsps, several antimicrobial genes, genes involved in the melanization process, and an antioxidant gene (PmSOD). PmHSF1 plays an important role in the thermal stress response, regulating the expression of Hsps and immune-related genes in P. monodon.

HSP70 and HSP90 are involved in shrimp Penaeus vannamei tolerance to AHPND-causing strain of Vibrio parahaemolyticus after non-lethal heat shock.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus carrying toxin-producing plasmid, has led to severe mortalities in farmed penaeid shrimp throughout Asia. Previous studies reported that a non-lethal heat shock (NLHS) could enhance disease tolerance in aquatic animals. Here, we investigate whether the NLHS could enhance the survival of shrimp Penaeusvannamei upon challenge with an AHPND-causing strain of V. Parahaemolyticus (VPAHPND). Two NLHS conditions, acute and chronic NLHSs, were used. The former abruptly exposed the juveniles shrimp from 28 °C to 38 °C for 30 min only once whereas the latter exposed the shrimp to 38 °C for 5 min every day for 7 days. The treated shrimp were, then, challenged with VPAHPND at day 3, day 7, and day 30 during the recovery time after the treatment. The results showed that the shrimp exposed to either acute or chronic NLHS had higher survival rate (>50%) than that of the non-heated shrimp control (20%) when they were challenged with VPAHPND at day 3 recovery time. However, only those exposed to chronic NLHS showed the VPAHPND protection at day 7 and day 30 recovery times. Furthermore, the qRT-PCR analysis revealed that the expression of heat shock proteins, LvHSP70, LvHSP90 as well as other immune-related genes, LvproPO and LvCrustin, were induced upon exposure of shrimp to chronic NLHS. Interestingly, gene silencing of LvHSP70 and LvHSP90 eliminated the VPAHPND tolerance in the chronic NLHS shrimp and had decreasing PO activity suggesting that these LvHSPs played crucial roles in bacterial defense in shrimp. All together, we show for the first time that the NLHS enhance the shrimp tolerance to VPAHPND infection and this is likely mediated by the induction of LvHSP70, LvHSP90 and subsequent activation of the proPO system.