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.

Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2) Bcl-2 in the orange-spotted grouper (Epinephelus coioides).

Bcl-2 is a pro-survival member of Bcl-2 like superfamily, playing an important role in regulating the apoptotic process. In this study, the full-length Bcl-2 (EcBcl-2) was obtained, consisting of a 5'UTR of 290 bp, an ORF of 699 bp and a 3'UTR of 920 bp. EcBcl-2 gene encoded a polypeptide of 232 amino acids with an estimated molecular mass of 26.12 KDa and a predicted isoelectric point (pI) of 6.93. The deduced amino acid sequence analysis showed that EcBcl-2 consisted of the conserved residues and characteristic domains known to the critical functionality for Bcl-2. qRT-PCR analysis revealed that EcBcl-2 transcript was expressed in all the examined tissues, while the strongest expression level was observed in liver, followed by the expression in blood, gill, kidney, spleen, heart, intestine and muscle. The groupers challenged with V. alginolyticus showed a significant increase of EcBcl-2 mRNA in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcBcl-2 protein expression was detected in liver. Subcellular localization analysis revealed that EcBcl-2 was localized in both nucleus and cytoplasm. Overexpression of EcBcl-2 can inhibit the LPS-induced apoptosis and activate the transcription activity of NF-κB and AP-1, while the deletion of BH1, BH2, BH3 or BH4 domain from EcBcl-2 can impede the signaling transduction. These results indicate that EcBcl-2 may play a regulatory role in the apoptotic process.

Molecular cloning, characterization and function of a germinal center kinase MST4 gene from Litopenaeus vannamei in response to Vibrio alginolyticus challenge in TLR-TRAF6 signaling pathway.

The serine/threonine protein kinase MST4 plays multiple roles in the regulation of signaling pathways that govern cellular processes including mitosis, migration, homeostasis, polarity, proliferation, differentiation and apoptosis. Here we report the identification and characterization of the full-length sequence of LvMST4 from the shrimp L. vannamei, and investigations into its role in the shrimp's immune response to infection by the pathogenic bacterium Vibrio alginolyticus. Subcellular localization assays demonstrated the enzyme's presence in the shrimp's cytoplasm, and tissue-specific expression analysis revealed that it is expressed ubiquitously but at different levels in different tissues. Infection with V. alginolyticus increased LvMST4 expression and induced a rapid response via the TLR-TRAF6 signaling pathway, causing a decline in the total hemocyte count (THC) and an increase in respiratory burst (RB) activity. In non-infected shrimp, RNAi silencing of LvMST4 with dsRNA had no significant effect on THC but seemed to activate the TRAF6-MKK6-p38 pathway and reduced RB activity. In shrimp challenged with V. alginolyticus, LvMST4 silencing reduced bacterial clearance and increased the initial upregulation of LvTRAF6 while reducing the expression of LvMKK6 and Lvp38. LvMST4 silencing also slightly reduced the THC but caused pronounced increases in RB activity and cumulative mortality. These findings suggest that LvMST4 contributes to antimicrobial responses via the TLR-TRAF6 signal pathway, and helps maintain immunological homeostasis in L. vannamei.

Molecular characterization and function of the Prohibitin2 gene in Litopenaeus vannamei responses to Vibrio alginolyticus.

Prohibitin2 (PHB2), a potential tumor suppressor protein, plays important roles in inhibition of cell cycle progression, transcriptional regulation, apoptosis and the mitochondrial respiratory chain. To explore its potential roles in crustaceans' immune responses we have identified and characterized LvPHB2, a 891 bp gene encoding a 297 amino acids protein in the shrimp Litopenaeus vannamei. Expression analyses showed that LvPHB2 is expressed in all examined tissues, and largely present in cytoplasm, correlating with its known anti-oxidation function in mitochondria. Luciferase reporter assays showed that over-expression of LvPHB2 could activate the p53 pathway, indicating that it might participate in apoptosis regulation. Quantitative real-time PCR revealed that infection with Vibrio alginolyticus induces its up-regulation in hepatopancreas. Moreover, RNAi knock-down of LvPHB2 in vivo raises mortality rates of L. vannamei infected by V. alginolyticus, and affects expression of STAT3, Caspase3 and p53 genes. We found significantly higher reactive oxygen species production, DNA damage and apoptosis rates in LvPHB2-silenced shrimp challenged with V. alginolyticus than in controls injected with a Green Fluorescent Protein-silencing construct. Our results suggest that LvPHB2 plays a vital role in shrimp responses to V. alginolyticus infection through its participation in regulation of oxidants and apoptosis.

Molecular cloning and characterization of PTEN in the orange-spotted grouper (Epinephelus coioides).

PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process.

Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2 associated X protein) Bax in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge.

Bax is a pro-apoptotic member of Bcl-2 like superfamily, playing an important role in regulating the apoptosis. In this study, the full-length Bax (EcBax) was obtained, containing a 5'UTR of 64 bp, an ORF of 579 bp and a 3'UTR of 1021 bp. The EcBax gene encoded a polypeptide of 192 amino acids with an estimated molecular mass of 21.55 KDa and a predicted isoelectric point (pI) of 6.75. The deduced amino acid sequence analysis showed that EcBax comprised the conserved residues and the characteristic domains known to the critical function of Bax. qRT-PCR analysis revealed that EcBax mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in blood, followed by the expression in liver, gill, spleen, kidney, heart, muscle and intestine. A sharp increase of EcBax expression was observed in the vibrio challenge group by comparing with those in the control. Subcellular localization analysis revealed that EcBax was predominantly localized in the cytoplasm. EcBax exerted a regulatory role in modulating the mitochondrial membrane potential, promoting the cytochrome c release, and then activating the downstream caspase signaling. Moreover, the overexpression of EcBax can decrease the cell viability and antagonize NF-kB, AP-1, Stat3 promoter activity in Hela cells. These results indicate that EcBax containing the conserved domain of pro-apoptotic member of Bcl-2 family may disrupt the mammalian signaling and play a regulative role in the apoptotic process.