[Show abstract][Hide abstract] ABSTRACT: Many viruses can hijack the host cell NF-κB as part of their life cycle, diverting NF-κB immune regulatory functions to favor their replications. There were several reports on the functions of Litopenaeus vannamei NF-κB (LvNF-κB) in White spot syndrome virus (WSSV) replication in vitro. Here, we studied the relationship between LvNF-κB family protein Dorsal (LvDorsal) and Relish (LvRelish) with WSSV replication in vivo. The expressions of LvDorsal and LvRelish were significantly upregulated by WSSV challenge. Virus loads and expression of viral envelope protein VP28 in LvDorsal or LvRelish silencing shrimps were significantly lower than the control shrimps injected with EGFP-dsRNA or PBS after challenge with 1x10(5) copies WSSV/shrimp. In addition to the LvDorsal activation of WSV069 and WSV303 promoter that we have reported, LvRelish can also activate WSV069 and WSV303 promoter by dual luciferase reporter assays through screening 40 WSSV gene promoters that have putative multiple NF-kB binding sites. The promoter activity of the WSV069 by LvDorsal activation was significantly higher than that by LvRelish activation. WSSV replication in LvDorsal, LvRelish or WSV303 silencing shrimps were significantly inhibited. These results indicate that the L vannamei NF-κB family proteins LvDorsal and LvRelish expressions are significantly activated by WSSV challenge and WSSV replication partially relied on the activations of LvDorsal and LvRelish in vivo.
Full-text · Article · Mar 2014 · Developmental and comparative immunology
[Show abstract][Hide abstract] ABSTRACT: Heat shock transcription factors belong to the heat shock factor (HSF) protein family, which are involved in heat shock protein (HSP) gene regulation. They are critical for cell survival upon exposure to harmful conditions. In this study, we identified and characterized a HSF1 (LvHSF1) gene in Litopenaeus vannamei, with a full-length cDNA of 2841 bp and an open reading frame encoding a putative protein of 632 amino acids. Through multiple sequence alignment and phylogenetic analysis, it was revealed that LvHSF1 was closed to insect HSF family, which contained a highly conserved DNA-binding domain, oligomerization domains with HR-A/B, and a nuclear localization signal. Tissues distribution showed that LvHSF1 was widely expressed in all tissues tested. And it was upregulated in hemocytes and gills after Vibrio alginolyticus or Staphylococccus aureus infection. Dual-luciferase reporter assays indicated that LvHSF1 activated the promoters of L. vannamei HSP70 (LvHSP70) and L.vannamei Cactus (LvCactus), while inhibited the expressions of Drosophila antimicrobial peptide (AMP) Atta, Mtk, and L. vannamei AMP PEN4 through NF-κB signal transduction pathway modification. Knocked-down expression of LvHSF1 by dsRNA resulted in downregulations of LvHSP70 and LvCactus, as well as cumulative mortality decreasing under V. alginolyticus or S. aureus infection in L. vannamei. Taken together, our data strongly suggest that LvHSF1 is involved in LvHSP70 regulation, therefore plays a great role in stress resistance. And it also takes part in LvCactus/LvDorsal feedback regulatory pathway modification of L. vannamei, which is in favor of V. alginolyticus or S. aureus infection.
Full-text · Article · Feb 2014 · Fish & Shellfish Immunology
[Show abstract][Hide abstract] ABSTRACT: p38 mitogen-activated protein kinases (MAPKs) are broadly expressed from yeasts to mammals, and are involved in the regulation of cells responsible to various extracellular stimuli. In this study, a p38 MAPK gene (designated as Lvp38) from Litopenaeus vannamei, was cloned and characterized. It contained the conserved structures of a Thr-Gly-Tyr (TGY) motif and a substrate-binding site, Ala-Thr-Arg-Trp (ATRW). The tissue distribution patterns showed that Lvp38 was widely expressed in all examined tissues, with the highest expression in hemocytes, nerves, and intestines. Quantitative real-time PCR revealed that Lvp38 was upregulated in gills and hemocytes after infection with the Gram-negative Vibrio alginolyticus and the Gram-positive Staphylococcus aureus. Reporter gene assays indicated that Lvp38 activated the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp. Knockdown of Lvp38 by RNA interference (RNAi) resulted in a higher mortality of L. vannamei under V. alginolyticus and S. aureus infection, as well as a reduction in the expression of three shrimp AMP genes, namely, PEN4, crustin, and ALF2. Taken together, our data indicated that Lvp38 played a role in defending against bacterial infections.
Full-text · Article · Mar 2013 · Fish & Shellfish Immunology
[Show abstract][Hide abstract] ABSTRACT: Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G) were identified in All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, , , and white spot syndrome virus (WSSV). Dual-luciferase reporter assays in Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of upon but not and WSSV infections. The expression of AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.
[Show abstract][Hide abstract] ABSTRACT: Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.
[Show abstract][Hide abstract] ABSTRACT: The RNA interference (RNAi) is an evolutionarily conserved protective mechanism in eukaryotes against parasitic foreign nucleic acids. Previous studies demonstrated that the RNAi mechanism is important for shrimp antiviral immunity. Here, we report the identification and functional analysis of two key components of the shrimp RNAi activity: Litopenaeus vannamei arsenite resistance gene 2 (LvArs2) and partner of drosha (LvPasha). The full-length cDNA of LvArs2 was 3470 bp, including a 5' untranslated region (UTR) of 167 bp, a 3' UTR of 639 bp, and an open reading frame (ORF) of 2664 bp that encoded 887 amino acid residues with an estimated molecular mass of 102.5 kDa. The full-length cDNA of LvPasha was 2654 bp, including a 5' UTR of 99 bp, a 3' UTR of 560 bp, and an ORF of 1995 bp that encoded 664 amino acid residues with an estimated molecular mass of 74.2 kDa. Co-immunoprecipitation demonstrated that LvArs2 interacted with L. vannamei Dicer2 (LvDcr2) and LvPasha in Drosophila Schneider 2 (S2) cells, suggesting that LvArs2 may be involved in regulation of the miRNA/siRNA pathways in L.vannamei. Subcellular localization assays demonstrated both LvArs2 and LvPasha proteins mainly presented in the nucleus. After Poly(C-G) stimulation, the expression of LvArs2 was suppressed and expression of LvPasha was enhanced in shrimp gills. These results suggest that LvArs2 and LvPasha may participate in the defense against RNA viruses in crustacea.
Full-text · Article · Dec 2011 · Fish & Shellfish Immunology