RNAi screening identifies a new Toll from shrimp Litopenaeus vannamei that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides.

The function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-κB transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins to inhibit viral infection. Taken together, we therefore identify that the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs.

Identification and characterization of transforming growth factor ß-activated kinase 1 from Litopenaeus vannamei involved in anti-bacterial host defense.

LvTAK1, a member of transforming growth factor ß-activated kinase 1 (TAK1) families, has been identified from Litopenaeus vannamei in this study. The full length of LvTAK1 is 2670 bp, including a 2277 bp open reading frame (ORF) that encoded a putative protein of 758 amino acids with a calculated molecular weight of ∼83.4 kDa LvTAK1 expression was most abundant in muscles and was up-regulated in gills after LPS, Vibrio parahaemolyticus, Staphylococcus aureus, Poly (I:C) and WSSV challenge. Both in vivo and in vitro experiments indicated that LvTAK1 could activate the expression of several antimicrobial peptide genes (AMPs). In addition, the dsRNA-mediated knockdown of LvTAK1 enhanced the susceptibility of shrimps to Vibrio parahaemolyticus, a kind of Gram-negative bacteria. These results suggested LvTAK1 played important roles in anti-bacterial infection. CoIP and subcellular localization assay demonstrated that LvTAK1 could interact with its binding protein LvTAB2, a key component of IMD pathway. Moreover, over-expression of LvTAK1 in Drosophila S2 cell could strongly induce the promoter activity of Diptericin (Dpt), a typical AMP which is used to read out of the activation of IMD pathway. These findings suggested that LvTAK1 could function as a component of IMD pathway. Interestingly, with the over-expression of LvTAK1 in S2 cell, the promoter activity of Metchnikowin (Mtk), a main target gene of Toll/Dif pathway, was up-regulated over 30 times, suggesting that LvTAK1 may also take part in signal transduction of the Toll pathway. In conclusion, we provided some evidences that the involvement of LvTAK1 in the regulation of both Toll and IMD pathways, as well as innate immune against bacterial infection in shrimp.

Interferon-Induced Protein 6-16 (IFI6-16) from Litopenaeus vannamei Regulate Antiviral Immunity via Apoptosis-Related Genes.

A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)-Vago-JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF-IFN-JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT-IFI6-16-caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp.

MKK4 from Litopenaeus vannamei is a regulator of p38 MAPK kinase and involved in anti-bacterial response.

LvMKK4, a homologue of the mammalian mitogen-activated protein kinase kinase 4 (MKK4), was isolated and identified from Litopenaeus vannamei in the present study. The full-length cDNA of LvMKK4 is 1947 bp long, with an open reading frame (ORF) of 1185 bp encoding a putative protein of 388 amino acids. LvMKK4 contains several characteristic domains such as D domain, SIAKT motif and kinase domain, all of which are conserved in MAP kinase kinase family. Like mammalian MKK4 but not Drosophila MKK4, LvMKK4 could bind to, phosphorylate and activate p38 MAPK, which provided some insights into the signal transduction mechanism of MKK4-p38 cascade in invertebrates. Our real-time PCR data indicated that LvMKK4 was ubiquitously expressed in all tested tissues and extraordinarily abundant in muscle. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvMKK4 activated the transcription of antimicrobial peptide genes (AMPs), including Drosophila Attacin A, Drosomycin, and shrimp Penaeidins. Additionally, LvMKK4 was up-regulated in both intestine and hepatopancreas by a variety of inflammatory stimuli including LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I: C) and white spot syndrome virus. Furthermore, RNAi-mediated knockdown of LvMKK4 enhanced the sensitivity of L. vannamei to V. parahaemolyticus infection. These findings suggested that LvMKK4 played an important role in anti-bacterial response and could be a potential target for inflammation treatment.

An invertebrate STING from shrimp activates an innate immune defense against bacterial infection.

It has been proposed that invertebrate stimulators of interferon genes (STINGs) do not take part in the innate immune response to infection. Herein, we identified a new STING homolog from pacific white shrimp Litopenaeus vannamei (LvSTING). Some amino acids crucial for recognizing cyclic dinucleotides in mammals are highly conserved in LvSTING. Moreover, LvSTING expression can be robustly induced by challenge with the Gram-negative bacteria Vibrio parahaemolyticus. Silencing of LvSTING contributes to decreased expression of the antimicrobial peptide PEN4 and renders shrimp more susceptible to V. parahaemolyticus infection, while coinjection with the recombinant LvSTING protein can rescue PEN4 expression in vivo and confer shrimp with more resistance to infection. Taken together, these results suggest that LvSTING is involved in the innate immune response to bacterial infection.

Identification and functional analysis of a TEP gene from a crustacean reveals its transcriptional regulation mediated by NF-κB and JNK pathways and its broad protective roles against multiple pathogens.

Thioester-containing proteins (TEPs) are present in a wide range of species from deuterostomes to protostomes and are thought to be involved in innate immunity. In the current study, a TEP gene homologous to insect TEPs (iTEP) from the crustacean Litopenaeus vannamei, named LvTEP1, is cloned and functionally characterized. The open reading frame (ORF) of LvTEP1 is 4383 bp in length, encoding a polypeptide of 1460 amino acids with a calculated molecular weight of 161.1 kDa LvTEP1, which is most similar to other TEPs from insects, contains some conserved sequence features, including a N-terminal signal peptide, a canonical thioester (TE) motif, and a C-terminal distinctive cysteine signature. LvTEP1 is expressed in most immune-related tissues, such as intestine, epithelium, and hemocytes, and the mRNA level of LvTEP1 is upregulated in hemocytes after bacterial and viral challenges, indicating its involvement in the shrimp innate immune response. An expression assay in Drosophila S2 cells shows LvTEP1 to be a full-length secretory protein, and processed forms are present in the supernatant. Of note, only the processed form of LvTEP1 protein can bind to both the gram-negative bacterium Vibrio parahaemolyticus and the gram-positive bacterium Staphylococcus aureus in vitro, and its abundance can be induced after bacterial treatment. Moreover, knockdown of LvTEP1 renders shrimps more susceptible to both V. parahaemolyticus and S. aureus, as well as white spot syndrome virus (WSSV) infection, suggesting its essential defensive role against these invading microbes. We also observe that the expression of LvTEP1 is regulated in a manner dependent on both NF-κB and AP-1 transcription factors in naive shrimps and in vitro, suggesting that LvTEP1 could be poised in the body cavity prior to infection and thus play an important role in basal immunity. Taken together, our findings provide some in vitro and in vivo evidence for the involvement of LvTEP1 in shrimp innate immunity and provide some insight into its expression regulation mediated by multiple transcription factors or signaling pathways.

Identification of two p53 isoforms from Litopenaeus vannamei and their interaction with NF-κB to induce distinct immune response.

p53 is a transcription factor with capability of regulating diverse NF-κB dependent biological progresses such as inflammation and host defense, but the actual mechanism remains unrevealed. Herein, we firstly identified two novel alternatively spliced isoforms of p53 from Litopenaeus vannamei (LvΔNp53 and the full-length of p53, LvFLp53). We then established that the two p53 isoforms exerted opposite effects on regulating NF-κB induced antimicrobial peptides (AMPs) and white spot syndrome virus (WSSV) immediate-early (IE) genes expression, suggesting there could be a crosstalk between p53 and NF-κB pathways. Of note, both of the two p53 isoforms could interact directly with LvDorsal, a shrimp homolog of NF-κB. In addition, the activation of NF-κB mediated by LvDorsal was provoked by LvΔNp53 but suppressed by LvFLp53, and the increased NF-κB activity conferred by LvΔNp53 can be attenuated by LvFLp53. Furthermore, silencing of LvFLp53 in shrimp caused higher mortalities and virus loads under WSSV infection, whereas LvΔNp53-knockdown shrimps exhibited an opposed RNAi phenotype. Taken together, these findings present here provided some novel insight into different roles of shrimp p53 isoforms in immune response, and some information for us to understand the regulatory crosstalk between p53 pathway and NF-κB pathway in invertebrates.

Shrimp TAB1 interacts with TAK1 and p38 and activates the host innate immune response to bacterial infection.

Mammalian TAB1 has been previously identified as transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) binding protein, which functions as the activator of TAK1 and p38. This report, for the first time, identified and characterized the homolog of TAB1 in shrimp, to be specific, the homolog gene from Litopenaeus vannamei, containing a 1560-bp open reading frame (ORF) that encoded a putative protein of 519 amino acids with the conserved PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain in N-terminal and a TAK1 binding motif in C-terminus, has been cloned and named LvTAB1. LvTAB1 was most abundant in gills and its expression could respond significantly to a series of stimuli, including LPS, Vibrio parahemolyticus and Staphylococcus aureus. Moreover, Co-immunoprecipitation (Co-IP) experiments showed that LvTAB1 could combine with LvTAK1 as well as Lvp38, two members of IMD-NF-κB/MAPK pathway, which meant LvTAB1 could have a role in regulating the activities of these kinases. Over-expression of LvTAB1 in drosophila S2 cells could improve the transcriptional levels of antimicrobial peptide genes (AMPs) such as Diptericin (Dpt), the hallmark of drosophila NF-κB activated genes, indicating its activation effect on NF-κB pathway. Furthermore, suppression of LvTAB1 expression in vivo by RNA-interference increased the sensibility of shrimps to V. parahaemolyticus infection, implying its protective role against bacterial infection. In conclusion, these results provide some insight into the function of LvTAB1 during bacterial infection.

MKK6 from pacific white shrimp Litopenaeus vannamei is responsive to bacterial and WSSV infection.

p38 mitogen-actived protein kinases (MAPKs) broadly exist from yeast to mammals and participate in diverse cellular responses to various stimuli, whose activation can be induced by the MAPK kinase 6 (MKK6). In this study, a novel MKK6 homolog from Litopenaeus vannamei (LvMKK6) was cloned and characterized. The transcript of LvMKK6 was 1465bp long with an open reading frame (ORF) of 987bp that encoded a polypeptide of 328 amino acids. LvMKK6 was a both cytoplasmic- and nuclear-localized protein and its expression was up-regulated with the treatment of different stimuli including LPS, Vibrio parahaemolyticus, Staphylococcus aureus, Poly (I:C) and white spot syndrome virus (WSSV). Overexpression of LvMKK6 could lead to activate the promoter activities of several antimicrobial peptides (AMPs) such as PEN4. The further investigation demonstrated that LvMKK6 could interact with and phosphorylate Lvp38, suggesting LvMKK6 was an activator of Lvp38. Knockdown of LvMKK6 caused attenuate expression of several AMPs and resulted in the higher mortality of shrimp under V. parahaemolyticus infection, suggesting LvMKK6 could play vital roles in defense against bacterial infection. Interestingly, silencing of LvMKK6 led to the lower virus loads and suppressed viral gene (VP28) expression during WSSV challenge. In addition, overexpression of LvMKK6 promoted the promoter activities of 19 WSSV immediate-early genes such as wsv069, wsv249, wsv108 and wsv403. These results suggested that LvMKK6 could be used by WSSV. Above all, these data provided experimental evidences that participation of LvMKK6 in regulating AMPs and host defense against bacteria, as well as the immune response to WSSV infection.

Shrimp with knockdown of LvSOCS2, a negative feedback loop regulator of JAK/STAT pathway in Litopenaeus vannamei, exhibit enhanced resistance against WSSV.

JAK/STAT pathway is one of cytokine signaling pathways and mediates diversity immune responses to protect host from viral infection. In this study, LvSOCS2, a member of suppressor of cytokine signaling (SOCS) families, has been cloned and identified from Litopenaeus vannamei. The full length of LvSOCS2 is 1601 bp, including an 1194 bp open reading frame (ORF) coding for a putative protein of 397 amino acids with a calculated molecular weight of ∼42.3 kDa. LvSOCS2 expression was most abundant in gills and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly (I: C) and white spot syndrome virus (WSSV). There are several STAT binding motifs presented in the proximal promoter region of LvSOCS2 and its expression was induced by LvJAK or LvSTAT protein in a dose dependent manner, suggesting LvSOCS2 could be the transcriptional target gene of JAK/STAT pathway. Moreover, the transcription of DmVir-1, a read out of the activation of JAK/STAT pathway in Drosophila, was promoted by LvJAK but inhibited by LvSOCS2, indicating that LvSOCS2 could be a negative regulator in this pathway and thus can form a negative feedback loop. Our previous study indicated that shrimp JAK/STAT pathway played a positive role against WSSV. In this study, RNAi-mediated knockdown of LvSOCS2 shrimps showed lower susceptibility to WSSV infection and caused lessened virus loads, which further demonstrated that the JAK/STAT pathway could function as an anti-viral immunity in shrimp.