LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection.

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Andrographolide promote the growth and immunity of Litopenaeus vannamei, and protects shrimps against Vibrio alginolyticus by regulating inflammation and apoptosis via a ROS-JNK dependent pathway.

Vibrio alginolyticus (V. alginolyticus) is one of the major pathogens causing mass mortality of shrimps worldwide, affecting energy metabolism, immune response and development of shrimps. In the context of the prohibition of antibiotics, it is necessary to develop a drug that can protect shrimp from V. alginolyticus. Andrographolide (hereinafter called Andr), a traditional drug used in Chinese medicine, which possesses diverse biological effects including anti-bacteria, antioxidant, immune regulation. In this study, we investigated the effect of Andr on growth, immunity, and resistance to V. alginolyticus infection of Litopenaeus vannamei (L. vannamei) and elucidate the underlying molecular mechanisms. Four diets were formulated by adding Andr at the dosage of 0 g/kg (Control), 0.5 g/kg, 1 g/kg, and 2 g/kg in the basal diet, respectively. Each diet was randomly fed to one group with three replicates of shrimps in a 4-week feeding trial. The results showed that dietary Andr improved the growth performance and non-specific immune function of shrimps. L. vannamei fed with Andr diets showed lower mortality after being challenged by V. alginolyticus. After 6 h of V. alginolyticus infection, reactive oxygen species (ROS) production, tissue injury, apoptosis, expression of inflammatory factors (IL-1 ß and TNFα) and apoptosis-related genes (Bax, caspase3 and p53) were increased in hemocytes and hepatopancreas, while feeding diet with 0.5 g/kg Andr could inhibit the increase. Considering that JNK are important mediators of apoptosis, we examined the influence of Andr on JNK activity during V. alginolyticus infection. We found that Andr inhibited JNK activation induced by V. alginolyticus infection on L. vannamei. The ROS scavenger N-acetyl-l-cysteine (NAC) suppressed V. alginolyticus-induced inflammation and apoptosis, suggesting that ROS play an important role in V. alginolyticus-induced inflammation and apoptosis. Treated cells with JNK specific activator anisomycin, the inflammation and apoptosis inhibited by Andr were counteracted. Collectively, Andr promote the growth and immunity of L. vannamei, and protects shrimps against V. alginolyticus by regulating inflammation and apoptosis via a ROS-JNK dependent pathway. These results improve the understanding of the pathogenesis of V. alginolyticus infection and provide clues to the development of effective drugs against V. alginolyticus.

MYC drives autophagy to adapt to stress in Penaeus vannamei.

MYC proto-oncogene (MYC), a first oncogenic nuclear transcription factor isolated from the human genome, belongs to the helix loop helix/leucine zipper protein family (bHLHzip). MYC plays an important part in the process of various physiological and biochemical of vertebrate, such as cell growth, proliferation, cycle, and autophagy. However, its molecular regulation mechanism and function in invertebrates are still unclear. In this study, a novel transcription factor MYC gene was screened, cloned, and characterized from Penaeus vannamei. The open reading frame of PvMYC was 1593bp, encode a polypeptide of 530 amino acids with molecular weight of 58.5 kDa, and a theoretical PI of 5.75. The results of tissue distribution showed that PvMYC was constitutively expressed in all detected tissues, and highest expression in hepatopancreas. The expression level of PvMYC up-regulated significantly and responded to low temperature stress by nuclear ectopic after low temperature stress. Overexpression of PvMYC in shrimp hemocytes negatively regulated the expression of Beclin-1 and reduced the conversion from LC3I to LC3II, yet p62 was decreased significantly. Meanwhile, RAPA eliminated the inhibition of autophagy caused by overexpression of PvMYC. ROS levels and autophagy flux showed the similar trend under low temperature stress after silencing PvMYC. The expression levels of Beclin-1, key ATG gene and LC3II increased significantly, while p62 decreased significantly under the same conditions. In addition, the Total hemocyte count (THC) decreased sharply, and accelerated the injury of hepatopancreas under low temperature stress after silencing PvMYC. Collectively, these results suggest that PvMYC has vital role in the cold adaptation mechanism of P. vannamei by negatively regulating autophagy.

Nemo-like kinase (NLK) gene regulates apoptosis via the p53 signaling pathway in Litopenaeus vannamei under low-temperature stress.

The Nemo-like kinase (NLK) is an important serine/threonine-protein kinase in many signaling pathways. However, its function in crustaceans, such as shrimps, is still poorly understood and needs to be further explored. In the present study, the full-length cDNA of NLK from Litopenaeus vannamei (LvNLK) was cloned. The full-length LvNLK cDNA has 2497 bp, including an open reading frame (ORF) of 1524 bp encoding a protein with 507 amino acids and a predicted molecular mass of 56.1 kDa. Phylogenetic analysis revealed that LvNLK shared high similarities with NLK from other known species. Low-temperature stress markedly upregulated the expression of LvNLK. Its overexpression in hemocytes suppressed the expression of BCL2-associated X (Bax) and tumor protein P53 (p53) in vitro. Meanwhile, the BCL2 apoptosis regulator (Bcl-2), MDM2 proto-oncogene (MDM2), and Yin Yang 1 (YY1) were upregulated. Moreover, LvNLK silencing in vivo increased the susceptibility of shrimps to low-temperature stress. The generation of ROS and the rate of hemocyte apoptosis also increased when LvNLK was silenced. Additionally, qPCR results indicated that LvNLK might participate in apoptosis via the p53 signaling pathway in vitro and in vivo. These results suggested that LvNLK is indispensable for the environmental adaptation of L. vannamei. Our current findings also demonstrated that NLK is evolutionarily conserved in crustaceans and provided insights into the environmental adaptation of invertebrates.

Transcriptome analysis of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas challenged by Vibrio alginolyticus reveals lipid metabolic disturbance.

Vibrio alginolyticus is a devastating bacterial pathogen of Pacific white shrimp (Litopenaeus vannamei), which often causes acute hepatopancreatic necrosis syndrome (AHPNS) and early mortality syndrome (EMS). Elucidation of molecular mechanisms of L. vannamei in responding to infection is essential for controlling the epidemic. In the present study, transcriptomic profiles of L. vannamei hepatopancreas were explored by injecting with PBS or V. alginolyticus. Hepatopancreas morphology of L. vannamei was also assessed. The result reveals that compared with the hepatopancreas of PBS group, the storage cells (R-cell), secretory cells (B-cell) and star-shaped polygonal structures of the lumen were disappeared and necrotic after challenged by V. alginolyticus at 24 h. Transcriptome data showed that a total of 314 differential expression genes were induced by V. alginolyticus, with 133 and 181 genes up- and down-regulated, respectively. These genes were mainly associated with lysosome pathway, glycerophospholipid metabolism, drug metabolism-other enzymes, cysteine and methionine metabolism, aminoacyl-tRNA biosynthesis and PPAR signal pathway. Among these pathways, the lysosome pathway, glycerophospholipid metabolism and PPAR signal pathway were both related with lipid metabolism. Therefore, we detected the lipid accumulation in hepatopancreas by Oil Red O staining, TG and CHOL detection and the relative mRNA expression of several lipid metabolism related genes in the hepatopancreas of shrimp after challenge to V. alginolyticus. The present data reveals that lipids from the L. vannamei are nutrient sources for the V. alginolyticus and define the fate of the infection by modulating lipid homeostasis. These findings may have important implication for understanding the L. vannamei and V. alginolyticus interactions, and provide a substantial dataset for further research and may deliver the basis for preventing the bacterial diseases.

TBC domain family 7-like enhances the tolerance of Penaeus vannamei to ammonia nitrogen by the up-regulation of autophagy.

TBC domain family 7 (TBC1D7) is one of the subunits of tuberous sclerosis complex (TSC) and an important regulator of autophagosome biogenesis. However, the function of TBC1D7 is not fully understood in crustaceans. In the present study, TBC1D7 was identified from Penaeus vannamei. The complete coding sequence of PvTBC1D7 was of 960 bp encoding a predicted polypeptide of 319 amino acids with one conserved TBC domain, which shared high similarity with TBC1D7 of that other species. The mRNA of PvTBC1D7 was highly expressed in hemocyte and hepatopancreas, and the PvTBC1D7 protein was localized specifically in the cytoplasm of hemocyte of shrimp. Besides, PvTBC1D7 was co-localized with PvTSC1 in the cytoplasm of shrimp, indicating that there might existed a binding relationship between PvTBC1D7 and PvTSC1. During the ammonia nitrogen stress, the mRNA transcripts of PvTBC1D7 were significantly upregulated in hemocyte, hepatopancreas, and gill. Functionally, overexpression of PvTBC1D7 in vitro restored the inhibition to autophagy caused by chloroquine (CLQ) and increased the autophagy level, while the silencing of PvTBC1D7 could inhibit the autophagy. More importantly, after interfering with PvTBC1D7, the autophagy level decreased significantly both in hepatopancreas and hemocyte of P. vannamei, the mRNA expression of PvmTOR was increased remarkably with the significantly decrease of autophagy-related genes (PvATG12 and PvATG14). And the reduction of PvTBC1D7 remarkably exacerbated the damage of hepatopancreas, increased the accumulation of ROS, and reduced the survival proportion of shrimp under ammonia nitrogen stress. Altogether, these results indicated that PvTBC1D7 might positively regulate the autophagy by stabilizing the negative regulation of mTOR by TSC complex, reduce the oxidative stress damage and improve shrimp ammonia nitrogen tolerance.