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.

Wang S ，Yin B ，Li H ，Xiao B ，Lǚ K ，Feng C ，He J ，Li C ... -  《-》

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.

Li C ，Li H ，Xiao B ，Chen Y ，Wang S ，Lǚ K ，Yin B ，Li S ，He J ... -  《-》

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.

Li C ，Chai J ，Li H ，Zuo H ，Wang S ，Qiu W ，Weng S ，He J ，Xu X ... -  《-》

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.

Li C ，Li H ，Wang S ，Song X ，Zhang Z ，Qian Z ，Zuo H ，Xu X ，Weng S ，He J ... -  《-》

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-β (TGF-β)-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.

Wang S ，Li M ，Yin B ，Li H ，Xiao B ，Lǚ K ，Huang Z ，Li S ，He J ，Li C ... -  《-》