Identification, Characterization, and Function Analysis of the Cactus Gene from Litopenaeus vannamei

Abstract

The nuclear factor-kappa B (NF-κB) pathways play important roles in innate immune responses. IκB is the main cytoplasmic inhibitor of NF-κB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IκB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-κB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-κB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV) infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.

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

“…Genome walking libraries were constructed by GenomeWalker™ Universal Kit (Clontech, Japan) according to the previous study [23]. The first round of genome walking PCR amplifications were performed using the primer pair of AP1/5 0 GW-LvJAK-1.…”

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

“…It has been known that NF-kB can upregulate the expression of IkB, and thus in turn attenuate the activation of NFkB itself (58). In L. vannamei, solid evidence has also supported that Dorsal could bind the promoter of Cactus to enhance its expression, thus constituting a negative feedback loop to regulate Dorsal activity (19). In this study, we also observed that the mRNA level of Cactus was increased after the miR-1959 mimics injection and decreased after the inhibitors injection.…”

“…At present, the main components of the TLR/NF-kB pathway, including three TLRs, two Tubes (homolog of IRAK4), Pelle (homolog of IRAK1), Myd88, TRAF6, Dorsal (homolog of NF-kB), Cactus (homolog of IkB), and so on, have been systemically studied in L. vannamei. This provides a basis for further investigation of the regulatory mechanism of shrimp immunity and establishes L. vannamei as a useful platform for studying the invertebrate NF-kB pathway (16)(17)(18)(19)(20).…”

“…Confocal laser scanning microscopy S2 cells were cotransfected with Dorsal-GFP expression vector (19), pAC5.1-Cactus/pAC5.1-Cactus-Mut and agomiR-1959/agomiR-NC, and 24 h later were treated with 0.5 mg/ml LPS (Sigma). After 6 h, the nuclear translocation rate of Dorsal was measured using a confocal laser scanning microscope (Leica, Germany) following a previously described method (19).…”

“…After 6 h, the nuclear translocation rate of Dorsal was measured using a confocal laser scanning microscope (Leica, Germany) following a previously described method (19).…”