A Novel Kelch-Like-1 Is Involved in Antioxidant Response by Regulating Antioxidant Enzyme System in Penaeus vannamei.

Heavy metals are typical cumulative pollutants that can enter and poison the human body through the food chain. However, the molecular mechanism of heavy metal-induced oxidative stress is unclear. In this study, we characterize PvKelch-like-1 from P. vannamei and explore its antioxidant roles in immune regulation of crustaceans. PvKelch-like-1 full length contains 2107 nucleotides, consists of a 5' untranslated region (UTR) of 79 bp, a 3' UTR of 180 bp, and a ORF of 1848 encoded 615 amino acids, which contain a BTB, BACK and Kelch motif, putative molecular mass and isoelectric point were 69 KDa and 6.54. PvKelch-like-1 mRNA was ubiquitously expressed in all detected tissue of P. vannamei, and mRNA expression levels were significantly up-regulated from 6 to 24 h after cadmium stress and reached the highest level (3.2-fold) at 12 h in the hepatopancreas. Subcellular localization analysis revealed that PvKelch-like-1 was localized in the nucleus. Silencing PvKelch-like-1 significantly increased reactive oxygen species (ROS) (1.61-fold) production and DNA damage (1.32-fold) in the shrimp hemolymph, and significantly decreased total hemocyte counts (THC) (0.64-fold) at 6 h in hemolymph. Additionally, the antioxidant genes PvCAT (0.43-fold), PvMnSOD (0.72-fold), PvGST (0.31-fold) and PvGPx (0.59-fold) at 6 h were decreased significantly in PvKelch-like-1 silenced shrimp after cadmium stress. Overexpression of PvKelch-like-1 has the opposite results in enzyme activity. The SOD (2.44-fold) and CAT (2.19-fold) activities were significantly increased after overexpressing PvKelch-like-1. These results suggest that PvKelch-like-1 plays a vital role in shrimp innate immune defense by positively regulating the expression of antioxidant enzyme genes to respond to cadmium stress.

Molecular characterization and function analysis of a nucleotide excision repair gene Rad23 from Litopenaeus vannamei after Vibrio alginolyticus challenge.

Nucleotide excision repair (NER) removes many different types of DNA lesions, and NER related host factors are reported to aid recovery steps during viral integration. Here, we report the identification and characterization of a DNA repair gene Rad23 from Litopenaeus vannamei and explore its role in innate immunity of crustaceans. LvRad23 contains a1149 bp open reading frame (ORF) which encodes a 382 amino acids protein with predicted theoretical isoelectric point of 4.21. LvRad23 was ubiquitously expressed in the muscle, eyestalk, gill, stomach, heart, legs, intestine, and hepatopancreas in order from high to low and LvRad23 protein was showed to be located in the cytoplasm of Drosophila S2 cells. The homology analysis showed that it has a high sequence homology with Rad23 protein from Marsupenaeus japonicus. Vibrio alginolyticus challenge induced a remarkable up-regulation of LvRad23 mRNA in hepatopancreas. Knocking down LvRad23can interfere the NER pathway by down regulating the expression of replication protein A (RPA) and proliferating cell nuclear antigen (PCNA). However it didn't cause any significant difference on total hemocyte count (THC) between LvRad23-silenced and non-silenced group.LvRad23-silenced then challenge with V. alginolyticus inducing high level of reactive oxygen species (ROS) and DNA damage in hemolymph. As well as decreased THC, which seriously diminished the innate immune system of L. vannamei. Meanwhile, the NER pathway was reactived by enhancing the expression of LvRad23 and promoting the production of LvPCNA to resist apoptosis and maintain proliferation of hemolymph cells in the later stage. Our results suggest that LvRad23 plays a vital role in shrimp specific immune response to V. alginolytcus through its participation in NER pathway.

Identifying the function of LvPI3K during the pathogenic infection of Litopenaeus vannamei by Vibrio alginolyticus.

It is well known that PI3K regulates various processes in mammalian cells by generating a secondary messenger that later activates AKT. However, its innate immune function in crustaceans remains unclear. We report the characterization of Litopenaeus vannamei PI3K (LvPI3K) for investigating how PI3K participates in the innate immunity of crustaceans. Full-length LvPI3K cDNA was 3357 bp long, with a 3222 bp open reading frame (ORF) that encodes a putative protein of 1292 amino acids. The PI3K catalytic domain (PI3Kc) of LvPI3K was found to be rather conserved when the PI3Ks from other species were analyzed. The LvPI3K protein was shown to be localized to the cytoplasm of Drosophila S2 cells, while LvPI3K mRNA was ubiquitously expressed in healthy L. vannamei, with the highest expression found in hemolymph. A dual luciferase reporter gene assay demonstrated that LvPI3K overexpression activated the promoter of antibacterial peptide LvPEN4 in a dose-dependent manner. However, the addition of PDTC, a specific inhibitor of NF-κB, suppressed the LvPI3K-induced LvPEN4 promoter activation. Moreover, Vibrio alginolyticus challenge induced a rapid up-regulation of LvPI3K expression. Further experiments showed that LvPI3K silencing in shrimp challenged with V. alginolyticus significantly increased Vibrio number, ROS production and DNA damage in the hemolymph, as well as significantly decreased total hemocyte count. The mRNA levels of certain molecules related to LvPI3K signaling, such as LvAKT and LvPEN4, also decreased following LvPI3K silencing. Taken together, these results suggest that LvPI3K regulates the downstream signal component LvPEN4 and functions in V. alginolyticus resistance.

LvCdc42 is a potential negative regulator of Lvp53 in Litopenaeus vannamei exposed to Vibrio alginolyticus stress.

As a crucial molecular switch, Cdc42 is a signal regulation hub which is involved in a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression and apoptosis. It has been reported that this GTPase promotes host defense against fatal infection and plays a vital role in the innate immunity system of mammals. But whether and how Cdc42 participates in innate immunity in invertebrates, such as the shrimp Litopenaeus vannamei, is still unknown. In this study, confocal microscopy analysis showed that LvCdc42 located in both cytoplasm and nucleus of S2 cells depended on its structure. The silencing LvCdc42 induced an increase in the expression of Lvp53 and Lvcaspase-3. When LvCdc42-silenced shrimps were stressed with Vibrio alginolyticus, the expression of Lvp53 and Lvcaspase-3 was markedly up-regulated. Moreover, the increase in the apoptosis rate in hemocytes and in cumulative mortality were in line with Lvp53 mRNA expression. These data suggest that the molecular switch LvCdc42 acts as a negative regulator of Lvp53 and participates in the apoptosis of hemocytes when L. vannamei is challenged with V. alginolyticus.