Hongliang Zuo

Presence of Tube isoforms in Litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway.

The toll-like receptor (TLR)/NF-κB signaling pathways play critical roles in the innate immune system. The intracellular signal transduction of most TLR pathways in invertebrate cells is triggered by formation of a heterotrimeric complex composed of MyD88, Tube and Pelle. In this study, we identified a Litopenaeus vannamei Pelle (LvPelle) and an isoform of L. vannamei Tube (LvTube) designated as LvTube-1. The interactions among LvPelle, LvTube/LvTube-1 and LvMyD88/LvMyD88-1 were elucidated and their functions during pathogen infections were investigated. Knockdowns of LvPelle and LvTube/LvTube-1 using RNAi strategy led to higher mortalities of shrimps during Vibrio parahemolyticus infection, and could reduce the genome copy number of white spot syndrome virus (WSSV) in the infected muscle tissue but did not affect the mortality caused by WSSV infection. The effects of LvPelle and LvTube/LvTube-1 on promoters containing NF-κB binding motifs were analyzed by dual-luciferase reporter assays and the results demonstrated that LvTube-1 could activate the NF-κB activity to significantly higher level than LvTube did. Moreover, tissue distributions of LvTube and LvTube-1 mRNAs and their expression profiles during pathogen and immune stimulant challenges were different, indicating that they could play different roles in immune responses. This is the first report of Tube isoforms in invertebrates. Together with our previous study on LvMyD88 isoforms, our results suggest that various isoforms of adaptor components may be involved in various regulatory patterns of signal transduction in invertebrate TLR/NF-κB pathway and this could be a strategy adopted by invertebrates to modulate immune responses.

A MicroRNA-Mediated Positive Feedback Regulatory Loop of the NF-κB Pathway in Litopenaeus vannamei

In the evolutionarily conserved canonical NF-κB pathway, degradation of the NF-κB inhibitor IκB in the cytoplasmic NF-κB/IκB complex allows the liberated NF-κB to translocate into the nucleus to activate various target genes. The regulatory mechanism governing this process needs further investigation. In this study, a novel microRNA, temporarily named miR-1959, was first identified from an invertebrate Litopenaeus vannamei. miR-1959 targets the 3′-untranslated region of the IκB homolog Cactus gene and reduces the protein level of Cactus in vivo, whereas the NF-κB homolog Dorsal directly binds the miR-1959 promoter to activate its transcription. Therefore, miR-1959 mediates a positive feedback regulatory loop, in that Dorsal activates miR-1959 expression, and in turn, miR-1959 inhibits the expression of Cactus, further leading to enhanced activation of Dorsal. Moreover, miR-1959 regulates the expression of many antimicrobial peptides in vivo and is involved in antibacterial immunity. To our knowledge, it is the first discovery of a microRNA-mediated feedback loop that directly regulates the NF-κB/IκB complex. This positive feedback loop could collaborate with the known NF-κB/IκB negative loop to generate a dynamic balance to regulate the activity of NF-κB, thus constituting an effective regulatory mechanism at the critical node of the NF-κB pathway.

Pellino protein from pacific white shrimp Litopenaeus vannamei positively regulates NF-κB activation.

Pellino, named after its property that binds Pelle (the Drosophila melanogaster homolog of IRAK1), is a highly conserved E3 class ubiquitin ligase in both vertebrates and invertebrates. Pellino interacts with phosphorylated IRAK1, causing polyubiquitination of IRAK1, and plays a critical upstream role in the toll-like receptor (TLR) pathway. In this study, we firstly cloned and identified a crustacean Pellino from pacific white shrimp Litopenaeus vannamei (LvPellino). LvPellino contains a putative N-terminal forkhead-associated (FHA) domain and a C-terminal ring finger (RING) domain with a potential E3 ubiquitin-protein ligase activity, and shows a high similarity with D. melanogaster Pellino. LvPellino could interact with L. vannamei Pelle (LvPelle) and over-expression of LvPellino could increase the activity of LvDorsal (a L. vannamei homolog of NF-κB) on promoters containing NF-κB binding motifs and enhance the expression of arthropod antimicrobial peptides (AMPs). The LvPellino protein was located in the cytoplasm and nucleus and LvPellino mRNA was detected in all the tissues examined and could be up-regulated after lipopolysaccharides, white spot syndrome virus (WSSV), Vibrio parahaemolyticus, and Staphylococcus aureus challenges, suggesting a stimulation response of LvPellino to bacterial and immune stimulant challenges. Knockdown of LvPellino in vivo could significantly decrease the expression of AMPs and increase the mortality of shrimps caused by V. parahaemolyticus challenge. However, suppression of the LvPellino expression could not change the mortality caused by WSSV infection, and dual-luciferase reporter assays demonstrated that over-expression of LvPellino could enhance the promoters of WSSV genes wsv069 (ie1), wsv303, and wsv371, indicating a complex role of LvPellino in WSSV pathogenesis and shrimp antiviral mechanisms.

A Janus Kinase in the JAK/STAT signaling pathway from Litopenaeus vannamei is involved in antiviral immune response.

The JAK/STAT signaling pathways are conserved in evolution and mediate diversity immune responses to virus infection. In the present study, a Janus kinase (designated as LvJAK) gene was cloned and characterized from Litopenaeus vannamei. LvJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7 and showed 19% identity (34% similarity) and 21% identity (35% similarity) to Drosophila Hopscotch protein and Human JAK2 protein, respectively. The mRNA of LvJAK was highly expressed in hepatopancreas of L. vannamei and its expression level was prominently upregulated after the stimulation of Poly (I:C) and white spot syndrome virus (WSSV) challenges. There were 10 putative STAT binding motifs in the promoter region of LvJAK, and it could be regulated by LvJAK self or (and) LvSTAT, suggesting that LvJAK is the JAK/STAT pathway target gene and could function as a positive regulator to form a positive feedback loop. In addition, the silencing of LvJAK caused higher mortality rate and virus load, suggesting that LvJAK could play an important role in defense against WSSV. This is the first report about the complete set of JAK/STAT proteins in shrimp and the results provide the evidence of the positive feedback loop mediated by JAK protein present in the JAK/STAT pathway in invertebrates.

The c-Fos and c-Jun from Litopenaeus vannamei play opposite roles in Vibrio parahaemolyticus and white spot syndrome virus infection

Growing evidence indicates that activator protein-1 (AP-1) plays a major role in stimulating the transcription of immune effector molecules in cellular response to an incredible array of stimuli, including growth factors, cytokines, cellular stresses and bacterial and viral infection. Here, we reported the isolation and characterization of a cDNA from Litopenaeus vannamei encoding the full-length c-Fos protein (named as Lvc-Fos). The predicted amino acid sequences of Lvc-Fos contained a basic-leucine zipper (bZIP) domain, which was characteristic of members of the AP-1 family. Immunoprecipitation and native-PAGE assays determined that Lvc-Fos could interact with the Lvc-Jun, a homolog of c-Jun family in L. vannamei, in a heterodimer manner. Further investigation demonstrated that Lvc-Fos and Lvc-Jun were expressed in all tested tissues and located in the nucleus. Real-time RT-PCR analysis showed both Lvc-Fos and Lvc-Jun in gills were up-regulated during Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges. In addition, reporter gene assays indicated Lvc-Fos and Lvc-Jun could activate the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp, as well as WSSV immediate early (IE) genes wsv069 and wsv249, in a different manner. Knockdown of Lvc-Fos or Lvc-Jun by RNA interference (RNAi) resulted in higher mortalities of L. vannamei after infection with V. parahaemolyticus, suggesting that Lvc-Fos and Lvc-Jun might play protective roles in bacterial infection. However, silencing of Lvc-Fos or Lvc-Jun in shrimp caused lower mortalities and virus loads under WSSV infection, suggesting that Lvc-Fos and Lvc-Jun could be engaged for WSSV replication and pathogenesis. In conclusion, our results provided experimental evidence and novel insight into the roles of L. vannamei AP-1 in bacterial and viral infection.

Identification and functional characterization of the TAB2 gene from Litopenaeus vannamei.

In Drosophila, TAB2, an important intermediate in the IMD signaling pathway, plays critical roles in the innate immune response in response to bacterial and viral infection. However, the role of TAB-related proteins in the immune response of shrimp has not yet been established. Here, we reported the identification of a TAB2-like gene in Litopenaeus vannamei designated as LvTAB2. The full-length cDNA of LvTAB2 was 2160 bp with an open reading frame of 1827 bp, which encoded a putative protein of 608 amino acids including a ubiquitin binding domain (CUE) at the N-terminal and a Zinc Finger domain (ZnF) at the C-terminus. Real-time RT-PCR analysis showed that LvTAB2 was expressed in all tested tissues and the expression levels of LvTAB2 in gills and hemocytes were positively induced in response to LPS, Vibrio parahemolyticus and White Spot Syndrome Virus (WSSV) challenges. Dual luciferase reporter assays demonstrated that LvTAB2 was able to induce the expression of antimicrobial peptide (AMP) genes, including Drosophila Attacin A and shrimp Penaeidins. Interestingly, over-expression of LvTAB2 could up-regulate the promoter activities of L. vannamei Vago1, Vago3 and Vago4 genes in S2 cells. To our knowledge, it was the first report that TAB2 participated in innate immune signaling to regulate the expression of Vago genes in invertebrates. Moreover, RNAi-mediated knockdown of LvTAB2 enhanced sensitivity of L. vannamei to Vibrio parahaemolyticus infection and caused elevated virus loads after WSSV infection. We suggested that the LvTAB2 may play important roles in the shrimp innate immunity.

Long-term influence of cyanobacterial bloom on the immune system of Litopenaeus vannamei.

Abstract Cyanobacteria are ubiquitously distributed in water on the Earth. It has long been known that the cyanobacterial bloom in aquaculture ponds can cause acute and massive deaths of shrimp. However, the long‐term and chronic effects of the cyanobacterial bloom on shrimp are still poorly understood. In this study, the immune state of white pacific shrimp, Litopenaeus vannamei, surviving a naturally occurring cyanobacterial bloom was investigated and tracked for 70 d. Compared with the control, the growth of shrimp suffering high concentrations of cyanobacteria was obviously postponed. In these shrimp, the activities of the NF‐&kgr;B, JAK/STAT and P38 MAPK immune signaling pathways and the expression of many antimicrobial peptide genes were down‐regulated, whereas the expression of C‐type lectins was significantly up‐regulated. Although the mRNA level of lysozyme was reduced, the expression of the invertebrate‐type lysozyme gene was increased. Furthermore, the concentration of hemocytes in hemolymph was greatly decreased, but the phagocytic activity of hemocytes was increased. These suggested that the cyanobacterial bloom has significant and complex influences on the immune system of shrimp, and in turn, alteration of the immune state could be a factor by which few shrimp can survive the cyanobacterial bloom. Thus, the current study could help further understand the interactions between the aquaculture water environment and the immune system of shrimp. HighlightsThe immune state of shrimp living in cyanobacterial bloom water was investigated.The NF‐&kgr;B, JAK‐STAT and P38 MAPK pathways were suppressed in shrimp under cyanobacterial stress.Expression of many antimicrobial peptides and lysozyme was down‐regulated.Expression of C‐type lectins and the invertebrate‐type lysozyme was up‐regulated.The concentration of hemocytes was reduced, but their phagocytic activity was increased.

Identification, characterization, and function analysis of the NF-κB repressing factor (NKRF) gene from Litopenaeus vannamei

Abstract The NF‐&kgr;B family transcription factors regulate a wide spectrum of biological processes, in particular immune responses. The studies in human suggest that the NF‐&kgr;B repressing factor (NKRF) negatively regulates the activity of NF‐&kgr;B through a direct protein–protein interaction. However, the function of NKRF has not been studied outside mammals up to now. The current study identified a NKRF gene (LvNKRF) from the Pacific white shrimp, Litopenaeus vannamei, which showed homology with NKRFs from insects, fishes and mammals. LvNKRF was high expressed in intestine, stomach and muscle tissues and was localized in the nucleus. LvNKRF could interact with both Dorsal and Relish, the two members of the shrimp NF‐&kgr;B family. Interestingly, although sharing a similar protein structure with that of human NKRF, LvNKRF showed no inhibitory but instead enhancing effects on activities of Dorsal and Relish, which was contrary to those of mammalian NKRFs. The expression of LvNKRF could not be induced by Gram‐positive and ‐negative bacteria and immunostimulants lipopolysaccharide (LPS) and poly (I:C) but was significantly up‐regulated after white spot syndrome virus (WSSV) infection. Silencing of LvNKRF significantly decreased the mortalities of shrimp caused by WSSV infection and down‐regulated the WSSV copies and the expression of WSSV structural gene in tissues. These suggested that LvNKRF could facilitate the infection of shrimp by WSSV, which may be an additional strategy for WSSV to hijack the host NF‐&kgr;B pathway to favor its own replication. The current study could provide a valuable context for further investigating the evolutionary derivation of NKRFs and facilitate the study of regulatory mechanisms of invertebrate NF‐&kgr;B pathways. HighlightsIdentification of the first invertebrate NKRF gene from Litopenaeus vannamei.LvNKRF exerts enhancing effects on activities of shrimp Dorsal and Relish.LvNKRF facilitates the replication of white spot syndrome virus in vivo.

Identification and characterization of an interleukin-16-like gene from pacific white shrimp Litopenaeus vannamei

&NA; Interleukins are a group of cytokines that play essential roles in immune regulation. Almost all interleukin genes are only found in vertebrates. In this study, an interleukin‐16‐like gene (LvIL‐16L) was identified from Pacific white shrimp, Litopenaeus vannamei. LvIL‐16L was predicted to encode a precursor (pro‐LvIL‐16L) with 1378 amino acids, sharing similarities with predicted pro‐IL‐16‐like proteins from insects. The C‐terminus of pro‐LvIL‐16L protein contained two PDZ domains homologous to the mature IL‐16 cytokine of vertebrates. In tissues, LvIL‐16L could be processed into a ˜36 kDa mature peptide through a caspase‐3 cleavage site, which was verified by in vitro site mutation analysis and in vivo RNA interference (RNAi) experiments. The LvIL‐16L mRNA could be detected in all the analyzed tissues and the expression of LvIL‐16L was significantly up‐regulated after immune stimulation. Using RNAi strategy, the role of LvIL‐16L in immune responses was initially investigated. Interestingly, knockdown of LvIL‐16L could significantly increase the mortality of the Vibro parahaemolyticus infected shrimps but reduce that of the WSSV infected shrimps, suggesting that LvIL‐16L could have opposite effects on the antiviral and antibacterial immune responses in shrimp. To our knowledge, this is the first study of an IL‐16‐like gene in invertebrates, which could help to elucidate interleukin evolution and regulatory mechanisms of shrimp immune responses. HighlightsStudy of the first invertebrate IL‐16 like gene (LvIL‐16L) from Litopenaeus vannamei.Identification of the mature LvIL‐16L peptide.LvIL‐16L plays different roles in antiviral and antibacterial immune responses.

Fatty acid synthase plays a positive role in shrimp immune responses against Vibrio parahaemolyticus infection.

ABSTRACT Fatty acid synthase (FAS) is an important enzyme that catalyzes the synthesis of fatty acids. In this study, the role of the FAS gene from pacific white shrimp Litopenaeus vannamei (LvFAS) in immune responses against Vibrio parahaemolyticus infection was studied. The expression of LvFAS could be up‐regulated upon infection of V. parahaemolyticus and stimulation of lipopolysaccharide and poly (I:C). The promoter of LvFAS was predicted to harbor a NF‐&kgr;B binding site and dual‐luciferase reporter assays demonstrated that the NF‐&kgr;B family proteins Relish, sRelish and Dorsal could activate the transcription of LvFAS. After knockdown of LvFAS expression using RNAi strategy, both the mortality of V. parahaemolyticus infected shrimps and the bacterial load in shrimp tissues were significantly increased. Meanwhile, the expression of many immune‐responsive genes, such as antimicrobial peptides, C‐type lectins (CTLs), lysozyme and hemolin, was down‐regulated. These suggested that LvFAS could play a positive role in anti‐V. parahaemolyticus responses in shrimp. To our knowledge, this is the first study that investigates the role of FAS in antibacterial immunity in animals, which may indicate the relationship between the anabolism of fatty acids and immune responses in crustaceans. HIGHLIGHTSExpression of LvFAS is up‐regulated during V. parahaemolyticus infection.The promoter of LvFAS is regulated by NF‐&kgr;B family proteins.Knockdown of LvFAS increases the mortality of V. parahaemolyticus infected shrimp.LvFAS influences expression of multiple immune‐responsive genes.