Antiviral phagocytosis is regulated by a novel Rab-dependent complex in shrimp penaeus japonicus

Rab GTPases are involved in phagosome formation and maturation. However, the role of Rab GTPases in phagocytosis against virus infection remains unknown. In this study, it was found that a Rab gene ( PjRab) from marine shrimp was upregulated in virus-resistant shrimp, suggesting that Rab GTPase was involved in the innate response to virus. The RNAi and mRNA assays revealed that the PjRab protein could regulate shrimp hemocytic phagocytosis through a protein complex consisting of the PjRab, beta-actin, tropomyosin, and envelope protein VP466 of shrimp white spot syndrome virus (WSSV). It was further demonstrated that the PjRab gene silencing by RNAi caused the increase in the number of WSSV copies, indicating that the PjRab might be an intracellular virus recognition protein employed by a host to increase the phagocytic activity. Therefore, our study presents a novel Rab-dependent signaling complex, in which the Rab GTPase might detect virus infection as an intracellular virus recognition protein and trigger downstream phagocytic defense against virus in crustacean for the first time. This discovery would improve our understanding of the still poorly understood molecular events involved in innate immune response against virus infection of invertebrates.     

Regulation of phagocytosis against bacterium by Rab GTPase in shrimp Marsupenaeus japonicus

Rab GTPases, members of the Ras superfamily, play important roles in phagosome formation and maturation. However, the involvement of Rab protein in phagocytosis against invading pathogens in crustacean remains unknown. In the present study, the RNAi and mRNA overexpression assays were conducted to elucidate the function of shrimp Rab gene (designated as PjRab) in hemocytic phagocytosis against bacterium. The results indicated that the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were significantly decreased when the PjRab gene was silenced by sequence-specific siRNA, suggesting that the PjRab protein was essential in hemocytic phagocytosis. On the other hand, the overexpression of PjRab gene leaded to the increase of phagocytic percentage and phagocytic index. The findings indicated that the PjRab protein was involved in the regulation of hemocytic phagocytosis of shrimp. Our report on the regulation of phagocytosis by Rab GTPase would contribute a better clue to realize the still poorly understood molecular events involved in shrimp as well as crustacean immune response.     

The role of white spot syndrome virus (WSSV) VP466 protein in shrimp antiviral phagocytosis

Widespread evidence indicates that the structural proteins of virus play very important roles in virus-host interactions. However, the effect of viral proteins on host immunity has not been addressed. Our previous studies revealed that the host shrimp Rab6 (termed as PjRab previously), tropomyosin, β-actin and the white spot syndrome virus (WSSV) envelope protein VP466 formed a complex. In this study, the VP466 protein was shown to be able to bind host Rab6 protein and increase its GTPase activity in vivo and vitro. Thus, VP466 could function as a GTPase-activating protein (GAP) of Rab6. In the VP466-Rab-actin pathway, the increase of the Rab6 activity induced rearrangements of the actin cytoskeleton, resulting in the formation of actin stress fibers which promoted the phagocytosis against virus. Therefore our findings revealed that a viral protein could be employed by host to initiate the host immunity, representing a novel molecular mechanism in the virus-host interaction. Our study would help to better understand the molecular events in immune response against virus infection in invertebrates.     

Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea)

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.     

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.     

Isolation and identification of novel microRNAs from Marsupenaeus japonicus

MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression. They play essential roles in various biological processes, such as development, differentiation and immune response. In this study, we identified 35 miRNAs from Marsupenaeus japonicus. Among them, fifteen miRNAs exhibited high homology to the known miRNAs from other arthropods, while the rest might represent novel miRNAs. We further showed a correlation of WSSV infection and the expression levels of 22 miRNAs. This is the first report to identify miRNAs from the shrimp. Our results extend the knowledge of the gene regulation of crustacean, providing clues for future researches of shrimp immunity against virus infection.     

RNA干扰抑制麻疹病毒体外复制的实验研究

为了研究短发夹RNA(shRNA)介导的RNA干扰对麻疹病毒体外复制的抑制作用,构建靶向与麻疹病毒复制密切相关的宿主细胞基因Rab9 GTPase基因特异性shRNA表达载体,分别转染Vero-E6和B95a细胞后感染麻疹病毒Edmonston株和野生株。逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western-blot)检测转染细胞内Rab9 GTPase基因表达水平;标准蚀斑试验测定麻疹病毒滴度。结果显示转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平同对照组相比明显降低,标准蚀斑试验显示麻疹病毒的复制受到显著抑制,抑制率达到90%以上。结果表明载体介导的shRNAs能通过特异性下调Rab9 GTPase基因表达抑制麻疹病毒体外复制,Rab9 GTPase可能成为治疗麻疹病毒感染的RNA干扰靶。     

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.     

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.     

Contribution of the caspase gene sequence diversification to the specifically antiviral defense in invertebrate

Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221-229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response.     

Disruption of Golgi morphology and trafficking in cells expressing mutant prenylated rab acceptor-1

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.     

Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double-stranded RNA

To determine whether Penaeus chinensis can be protected against white spot syndrome virus (WSSV) infection by intramuscular injection with long double-stranded RNAs (dsRNAs) as in other shrimp species and whether the protection degree by WSSV-specific dsRNAs is correlated with the roles of viral genes, P. chinensis juveniles were intramuscularly injected with long dsRNAs corresponding to VP28, VP281, protein kinase genes of WSSV, and an unrelated long dsRNA corresponding to a green fluorescence protein (GFP) gene. All shrimp injected with long dsRNAs including GFP dsRNA showed higher survival rates against WSSV infection than shrimp injected with PBS alone. Furthermore, shrimp injected with dsRNAs corresponding to VP28 and protein kinase showed higher survival rates than those injected with dsRNAs corresponding to VP281 and GFP. These results indicate that the introduction of long dsRNAs corresponding to viral proteins, which are essential for WSSV infection, is quite effective in blocking WSSV infection in P. chinensis, and suggest that dsRNA-mediated protection is a common feature across shrimp species.     

Rab6 Dependent Post‐Golgi Trafficking of HSV1 Envelope Proteins to Sites of Virus Envelopment

Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi‐to‐plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis. Rab6‐dependent virus production did not require the effectors myosin‐II, bicaudal‐D, dynactin‐1 or rabkinesin‐6, but was facilitated by ERC1, a factor involved in linking microtubules to the cell cortex. Tubulation and exocytosis of Rab6‐positive, glycoprotein‐containing membranes from the Golgi was substantially augmented by infection, resulting in enhanced and targeted delivery to cell tips. This reveals HSV1 morphogenesis as one of the first biological processes shown to be dependent on the exocytic activity of Rab6.      

Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture.     

Molecular cloning and expression pattern analysis of two novel disulfide isomerases in shrimp

Protein disulfide isomerase (PDI) catalyzes formation and isomerization of disulfide bridges and has chaperone activity. Currently, increasing evidence suggests the significance of PDI in immune and stress responses. To clarify the role of PDIs in the innate immunity of shrimp, two PDI genes were isolated and identified from Fenneropenaeus chinensis (fleshy prawn). FcPDI1 is 1878bp in length and encodes a protein of 383 amino acids. It has 18-amino acid signal peptide, 3 thioredoxin domains with 3 active sites of CGHC, and KEDL retention signal at its C-end. FcPDI1 is an atypical PDI. The open reading frame of FcPDI2 encodes a 497-amino acid protein and shows the classical domain organization a-b-b'-a'. Phylogenic analysis and multiple alignments show that FcPDI1 is similar to PDI that contains 3 thioredoxin domains from other species including invertebrates and vertebrates. FcPDI2, LvPDI, and insect PDIs are grouped into one cluster and are similar to PDIs having a-b-b'-a' domain organization. Tissue distribution shows that FcPDI1 and FcPDI2 were expressed in all detected tissues at the mRNA level. Changes in FcPDI1 and FcPDI2 expression at the mRNA level in hemocytes, hepatopancreas, gills, and ovaries upon Vibrio or white spot syndrome virus challenge were also analyzed. The results suggest that FcPDI1 and FcPDI2 might have roles in the innate immunity of shrimp. FcPDI1 was also successfully expressed in Escherichia coli and the recombinant FcPDI1 showed insulin reductase activity. Results show that FcPDI might play an important role in the innate immunity of shrimp.     

克氏原螯虾Rab5、Rab6蛋白及其抗体对血淋巴细胞吞噬活性的影响

为研究克氏原螯虾(Procambarus clarkii)Rab5(PcRab5)和Rab6(PcRab6)的生物学功能, 利用同源重组技术构建了克氏原螯虾pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体, 并进行了诱导表达和多克隆抗体的制备, 采用ELISA和Western Blot技术检测了抗体效价和特异性。将PcRab5和PcRab6的原核表达蛋白和多克隆抗体注射到健康克氏原螯虾体内, 研究了其对血淋巴细胞吞噬活性的影响。实验结果表明构建的pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体经诱导后可表达目的蛋白, 分子量均为67 kD, 纯化后蛋白条带单一, 纯度较高。重组表达纯化后的PcRab5和PcRab6蛋白免疫日本大耳兔分别获得效价为1:2048 K和1:512 K的兔抗血清, 制备的抗体可分别特异性识别PcRab5和PcRab6蛋白。将PcRab5和PcRab6蛋白注射健康克氏原螯虾后, 其血淋巴细胞中可吞噬荧光微球的细胞比例分别显著上升至38%和30%(P＜0.01)。而将纯化的PcRab5和PcRab6多克隆抗体分别注射至螯虾体内后, 其血淋巴细胞的吞噬细胞比例均出现显著下降(P＜0.05), PcRab5和PcRab6蛋白参与了血淋巴细胞吞噬功能。实验为进一步研究克氏原螯虾PcRab5和PcRab6分子功能奠定基础, 也可为理解甲壳动物Rab5和Rab6在血淋巴细胞吞噬功能中的作用提供帮助。     

Unique heparan sulfate from shrimp heads exhibits a strong inhibitory effect on infections by dengue virus and Japanese encephalitis virus

The structure and biological activities of a highly sulfated heparan sulfate (HS) extracted from shrimp (Penaeus brasiliensis) heads were characterized. Structurally the shrimp HS was more heterogenous than heparin, although it is still highly sulfated. The molecular mass of the shrimp HS preparation was determined to be 32.3 kDa by gel filtration HPLC. Analysis by surface plasmon resonance demonstrated that various growth/differentiation factors specifically bound to the shrimp HS with comparable affinity. Notably, the shrimp HS had a greater inhibitory effect against infections by dengue virus type 2 as well as Japanese encephalitis virus than heparin. Experiments on anticoagulant activity indicated that the shrimp HS exhibited significant anti-thrombin activity, but less than the commercial heparin. Hence, the HS preparation from shrimp heads, an industrial waste, is a prospective agent for a variety of clinical applications.