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.

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.