Regulation of lactate dehydrogenase in response to WSSV infection in the shrimp Litopenaeus vannamei.

Lactate dehydrogenase (LDH) is key for anaerobic glycolysis. LDH is induced by the hypoxia inducible factor -1 (HIF-1). HIF-1 induces genes involved in glucose metabolism and regulates cellular oxygen homeostasis. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive β-subunit (HIF-1β). The white spot syndrome virus (WSSV) induces anaerobic glycolysis in shrimp hemocytes, associated with lactate accumulation. Although infection and lactate production are associated, the LDH role in WSSV-infected shrimp has not been examined. In this work, the effects of HIF-1 silencing on the expression of two LDH subunits (LDHvan-1 and LDHvan-2) in shrimp infected with the WSSV were studied. HIF-1α transcripts increased in gills, hepatopancreas, and muscle after WSSV infection, while HIF-1β remained constitutively expressed. The expression for both LDH subunits increased in each tissue evaluated during the WSSV infection, translating into increased enzyme activity. Glucose concentration increased in each tissue evaluated, while lactate increased in gills and hepatopancreas, but not in muscle. Silencing of HIF-1α blocked the increase of LDH expression and enzyme activity, along with glucose (all tissues) and lactate (gills and hepatopancreas) concentrations produced by WSSV infection. These results demonstrate that HIF-1 up regulates the expression of LDH subunits during WSSV infection, and that this induction contributes to substrate metabolism in energetically active tissues of infected shrimp.

Hernández-Palomares MLE ，Godoy-Lugo JA ，Gómez-Jiménez S ，Gámez-Alejo LA ，Ortiz RM ，Muñoz-Valle JF ，Peregrino-Uriarte AB ，Yepiz-Plascencia G ，Rosas-Rodríguez JA ，Soñanez-Organis JG ... -  《-》

Hypoxia inducible factor -1 regulates WSSV-induced glycolytic genes in the white shrimp Litopenaeus vannamei.

Hypoxia-inducible factor -1 (HIF-1) is a transcriptional factor that regulates the expression of several glycolytic genes. The white spot syndrome virus (WSSV) induces a shift in glycolysis that favors viral replication in white shrimp Litopenaeus vannamei. HIF-1 is related to the pathogenesis of the WSSV infection through the induction of metabolic changes in infected white shrimp. Although the WSSV infection is associated with metabolic changes, the role of HIF-1 on key glycolytic genes during the WSSV infection has not been examined. In this work, we evaluated the effect of HIF-1α silencing on expression and activity of glycolytic enzymes (Hexokinase-HK, phosphofructokinase-PFK and pyruvate kinase-PK) along with the glucose transporter 1 (Glut1), regulatory enzymes (glucose-6-phosphate dehydrogenase-G6PDH and pyruvate dehydrogenase-PDH), and metabolic intermediates of glycolysis (glucose-6-phosphate-G6P and pyruvate). The expression of Glut1 increased in each tissue evaluated after WSSV infection, while HK, PFK and PK gene expression and enzyme activities increased in a tissue-specific manner. G6PDH activity increased during WSSV infection, and its substrate G6P decreased, while PDH activity decreased and its substrate pyruvate increased. Silencing of HIF-1α blocked the WSSV-induced Glut1 and glycolytic genes upregulation and enzyme activity in a tissue-specific manner. We conclude that HIF-1 regulates the WSSV-induced glycolysis through induction of glycolytic genes contributing to glucose metabolism in tissues of infected shrimp. Also, the inhibition, and activation of regulatory genes are likely to decrease the availability of the raw materials essential for WSSV replication and increase oxidative metabolism.

Godoy-Lugo JA ，Miranda-Cruz MM ，Rosas-Rodríguez JA ，Adan-Bante NP ，Icedo-García R ，Soñanez-Organis JG ... -  《-》

Characterization and role of PGK from Litopenaeus vannamei in WSSV infection.

Phosphoglycerate kinase (EC 2.7.2.3, PGK) catalyses the reversible transfer of a phosphate group from 1,3-diphosphoglyceric acid and ADP to produce 3-phosphoglyceric acid and ATP, which represents the initial production of ATP during glycolysis; therefore, PGK is a key enzyme in the energy metabolism. To study the role of PGK in the resistance to WSSV infection in shrimp, the full-length cDNA of the PGK gene (LvPGK) from Litopenaeus vannamei was obtained by using homology cloning and RACE amplification. The tissue distribution of LvPGK and its expression changes in the main immune tissues after WSSV stimulation were obtained by quantitative real-time PCR. Furthermore, RNA interference (RNAi) was used to study the role of LvPGK in shrimp defending against WSSV infection. The results showed that the full-length cDNA sequence of LvPGK was 1855 bp, contained a 1248 bp open reading frame (ORF) encoding 415 amino acids, and included a conserved PGK domain. LvPGK presented ubiquitous expression in most examined tissues, with the most predominant expression in the muscle and the weakest expression in the intestine. LvPGK transcripts could be induced in the hemocytes and hepatopancreas by injection with WSSV. Both the replication of WSSV and the shrimp cumulative mortality decreased significantly after LvPGK knockdown (P < 0.01). After challenging LvPGK RNAi shrimp with WSSV, the concentration of glucose in the hepatopancreas and muscle tissue did not show significant change; however, the content of pyruvate and lactate decreased significantly (P < 0.05). Moreover, significant decreases in the expression levels of crustin, ALF1, ALF2 and ALF3 were also detected. The results suggested that LvPGK might be involved in WSSV replication by increasing host aerobic and anaerobic metabolism.

Li FX ，Zhang YS ，Yao CL 《-》

Alternative splicing generates two lactate dehydrogenase subunits differentially expressed during hypoxia via HIF-1 in the shrimp Litopenaeus vannamei.

Soñanez-Organis JG ，Rodriguez-Armenta M ，Leal-Rubio B ，Peregrino-Uriarte AB ，Gómez-Jiménez S ，Yepiz-Plascencia G ... -  《-》

Glycogen phosphorylase of shrimp (Litopenaeus vannamei): Structure, expression and anti-WSSV function.

Glycogen phosphorylase (GP, EC 2.4.1.1) catalyze the rate-limiting step in glycogenolysis in animals, forming glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Therefore, GP plays a crucial role in carbohydrate metabolism. In the present study, the full-length cDNA sequence of GP (LvGP) was cloned from shrimp, Litopenaeus vannamei. The obtained 3242-bp LvGP cDNA sequence included a 5'-terminal untranslated region (UTR) of 48 bp, an open reading frame (ORF) of 2559 bp encoding a polypeptide of 852 amino acids (aa) and a 3'-UTR of 635 bp. The predicted LvGP protein sequence contained a typical phosphorylase domain (113-829 aa) and shared 72%-97% identities with GP from other species. Phylogenetic analysis revealed that LvGP showed the closest relationship with GP from Marsupenaeus japonicus. Tissue expression profiles showed that LvGP existed in most examined tissues, with the most predominant expression in the brain, followed by the muscles and stomach. LvGP transcripts in hepatopancreas and hemocytes were up regulated after the WSSV challenge. Furthermore, the role of LvGP in shrimp defending against WSSV infection was investigated by RNA interference (RNAi). Our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvGP RNAi (P < 0.01). The glycogen, glucose, and pyruvate content decreased in GP RNAi shrimp after WSSV injection, however, the lactate and ATP concentration enhanced. Moreover, lectin and anti-lipopolysaccharide factor2 (ALF2) were induced in LvGP silencing shrimp after WSSV infection, whereas the expression levels of crustin, ALF1 and ALF3 decreased. Our results suggested that the LvGP might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious gene expression.

Zhang YS ，Li FX ，Yao CL 《-》