RIP3 Associates with RIP1, TRIF, MAVS, and Also IRF3/7 in Host Innate Immune Signaling in Large Yellow Croaker Larimichthys crocea

Receptor-interacting protein 3 (RIP3) has been demonstrated to be a key regulator not only in cell death pathways including apoptosis and necroptosis but also in inflammation and host immune responses. In this study, a RIP3 ortholog named Lc-RIP3 is identified in large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of Lc-RIP3 is 1524 bp long and encodes a protein of 507 amino acids (aa). The deduced Lc-RIP3 protein has an N-terminal kinase domain and a C-terminal RHIM domain, and the genome organization of Lc-RIP3 is conserved in teleosts with 12 exons and 11 introns but is different from that in mammals, which comprises 10 exons and 9 introns. Confocal microscopy revealed that Lc-RIP3 is a cytosolic protein. The expression analysis at the mRNA level indicated that Lc-RIP3 is ubiquitously distributed in various tissues/organs, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Notably, Lc-RIP3 could induce NF-κB but not IRF3 activation. In addition, Lc-RIP3 co-expression with Lc-TRIF, Lc-MAVS, or Lc-IRF3 significantly abolishes the activation of NF-κB but enhances the induction of IRF3 activity. Moreover, NF-κB activity could be up-regulated when Lc-RIP3 is co-expressed with Lc-RIP1 or Lc-IRF7. These results collectively indicate that Lc-RIP3 acts as an important regulator in host innate immune signaling in teleosts.

The Effect of tonB Gene on the Virulence of Pseudomonas plecoglossicida and the Immune Response of Epinephelus coioides

Pseudomonas plecoglossicida is the causative agent of “visceral white spot disease” in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides. To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB-RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB-RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida. Meanwhile, the E. coioides infected with the tonB-RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB-RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB-RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.