Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

TLR4 involved in immune response against Vibrio Parahaemolyticus by MyD88-dependent pathway in Crassostrea hongkongensis.

Vibrio parahaemolyticus (V. parahaemolyticus) is a salt-loving gram-negative bacterium, and is the leading cause of mortality in cultured shellfish in recent years. Toll-like Receptor 4 (TLR4) is a classical pattern recognition receptor (PRRs) that recognizes pathogen-associated molecular patterns (PAMPs) of pathogenic microorganism and activates the immune response. However, the function and signal pathway of TLR4 in oyster are still unknown. In this study, a new TLR4 gene was identified from the Crassostrea hongkongensis (C. hongkongensis). The ChTLR4 contained an open reading frame of 2643 bp, encoding 880 amino acids with seven leucine-rich repeat (LRR) domains and a Toll/IL-1R (TIR) domain. The ChTLR4 shared the highest sequence identity (83.0%) with TLR4 of Crassostrea gigas. Tissue expression analysis revealed that ChTLR4 showed the highest constitutive expression in the gill and hepatopancreas, and was significantly upregulated in immune tissues post V. parahaemolyticus infection, especially in gill and hemocytes. Moreover, TLR4 silencing significantly inhibited the immune-enzyme activities, including SOD, CAT, ACP, AKP in gill and LZM in hemolymph supernatant, and increased MDA content in hemolymph supernatant. Meanwhile, the antimicrobial activities of the hemolymph supernatant were also significantly inhibited by TLR4 silencing. These data demonstrated that the ChTLR4 involved in innate immune response of C. hongkongensis against V. parahaemolyticus challenge. Finally, qRT-PCR analysis showed that ChTLR4 silencing clearly inhibited the expression of genes in TLR4-MyD88 pathway, indicating that MyD88-dependent pathway played a crucial role in ChTLR4-mediated immune response against V. parahaemolyticus.

Creb2 involved in innate immunity by activating PpMitf-mediated melanogenesis in Pteria penguin.

cAMP response element binding protein 2 (CREB2) acts as an intracellular transcriptional factor and regulates many physiological processes, including melanogenesis and melanocyte differentiation. In our previous research, the Creb2 gene has been characterized from Pteria penguin (P. penguin), but its role and regulatory mechanism in P. penguin are still unclear. In this study, first, the function of PpCreb2 in melanogenesis and innate immunity were identified. PpCreb2 silencing significantly decreased the tyrosinase activity and melanin content, indicating PpCreb2 played an important role in melanogenesis. Meanwhile, PpCreb2 silencing visibly suppressed the antibacterial activity of hemolymph supernatant, indicating that PpCreb2 was involved in innate immunity of P. penguin. Second, the PpCreb2 was confirmed to perform immune function by regulating the melanogenesis. The decreased melanin oxidation product due to PpCreb2 silencing triggered the declining of antibacterial activity of hemolymph supernatant, which then could be rescued by adding exogenous melanin oxidation products. Third, the regulation pathway of PpCreb2 involved in innate immunity was analyzed. The promoter sequence analysis of PpMitf discovered 5 conserved cAMP response element (CRE), which were specifically recognized by basic Leucine zipper domain (bZIP) of upstream activation transcription factor. The luciferase activities analysis showed that PpCreb2 could activate the CRE in PpMitf promoter via highly conserved bZIP domain and regulate the expression of PpMitf, which further regulated the PpTyr expression. Therefore, the results collectively demonstrated that PpCreb2 participated in innate immunity by activating PpMitf-mediated melanogenesis in P. penguin.