Nitric oxide synthase-dependent immune response against gram negative bacteria in a crustacean, Litopenaeus vannamei

Abstract

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus -NOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming.

Modulation of Crustacean Innate Immune Response by Amino Acids and Their Metabolites: Inferences From Other Species

“…When the Caribbean spiny lobster ( Panulirus argus ) and red swamp crayfish ( Procambarus clarkii ) were challenged with Escherichia coli or lipopolysaccharide (LPS), NO levels and the activity of NOS both increased, as part of the immune response ( 25 , 83 ). It has also been observed that when an NOS inhibitor or P. argus generated anti-NOS serum is administered to P. vannamei , it reduces bacterial clearance, further illustrating the importance of NOS in the immune defense of crustaceans ( 24 , 83 ).…”

“…When the Caribbean spiny lobster (Panulirus argus) and red swamp crayfish (Procambarus clarkii) were challenged with Escherichia coli or lipopolysaccharide (LPS), NO levels and the activity of NOS both increased, as part of the immune response (25,83). It has also been observed that when an NOS inhibitor or P. argus generated anti-NOS serum is administered to P. vannamei, it reduces bacterial clearance, further illustrating the importance of NOS in the immune defense of crustaceans (24,83). Met enhances leukocytes proliferation and reduces expression of pro-inflammatory genes (43) E. sinensis GSH supplementation promotes expression of immune genes (alf1 alf2 alf3, crus1, and crus2),, Arg kinase is one of the most important enzymes that regulates energy metabolism in invertebrates (84).…”

“…Polyamines are important Arg metabolites that have antitumor effect in mammals ( 75 , 76 ) and are also involved in the synthesis of T-cells ( 77 ). Similarly, NO, which is generated from Arg metabolism by the action of NOS, is an effective antibacterial agent against intracellular and extracellular pathogens ( 24 , 78 ). While NO has antimicrobial effects and also modulates immune response in host cells, excess NO levels could promote peroxynitrite synthesis to generate hydroxyl radicals that cause cell damage and/or cell death ( 20 , 79 ).…”

“…For instance, details are emerging on the pathogenicity and infection mechanism of WSSV, which requires glutamic acid for replication in shrimp ( 56 ). During Arg metabolism, increased activity of NOS has been shown to enhance functions of the immune system in P. vannamei , while Arg kinase promotes WSSV replication ( 24 , 26 ). This suggests that Arg might be the bridge between energy metabolism and immune response.…”

“…Role of amino acids and their metabolism in the immune response of crustaceans and other marine species. Increased NO and NOS mRNA levels to improve antibacterial immune response (23) P. vannamei; Panulirus argus Increased NO levels and NOS activity to improve antibacterial immune response(24,25) …”