Characterization of the crustin family of antimicrobial peptides in the Pacific white shrimp, Litopenaeus vannamei.

摘要

Antimicrobial peptides (AMPs) are key players in the innate immune response of all organisms, including crustaceans. Characterization of AMPs in shrimp provides an understanding of shrimp immunity to help prevent and control disease. For example, the crustins are constitutively expressed in great abundance in a subpopulation of hemocytes. In this study, the native crustin peptides and recombinantly produced crustins are characterized in their form and function. The objective is to determine how these peptides work as immune effectors in L. vannamei.;Native crustin was isolated and sequenced from L. vannamei hemocytes. After numerous efforts, it was deemed unfeasible to isolate enough peptide for antimicrobial and other functional assays. Recombinant expression of the carboxy-terminal unique (CTU) portion as well as the full length peptide was successful. However, recombinant peptides showed no consistent antimicrobial activity with any of the concentrations tested (up to 50muM) on any of the bacteria strains including Micrococcus luteus, Bacillus megaterium, Staphlococcus aureus, Escherichia coli, and Vibrio penaeicida .;Native crustin function was assessed by immune challenge after dsRNA knockdown, and depeletion of crustins caused increased mortality (up to 100%) in shrimp challenged with Vibrio penaeicida, but no significant change in mortality when infected with the fungus, Fusarium oxysporum . Crustins were localized in the hemocytes and in several tissue types in unchallenged shrimp as seen by immunocytochemistry. Endogenous crustins binding bacteria were also observed by bacteria affinity bead assays as well as immunocytochemistry.;Search of a mass EST collection from L. vannamei tissue libraries (eye stalk, gill, hemocytes, hepatopancreas, lymphoid organ, and nerve cord) for crustin sequence diversity provided identification of all known crustin types (I, II and III) as well as a fourth type. The Type IV crustins appear to be nerve cord specific, and the type I crustins are found primarily from gill tissue. A range of isoforms occurs within the specific types as well.;The results of this dissertation research demonstrate that crustins are involved in immune function in L. vannamei. And, although direct antimicrobial action was not seen with recombinant peptides, the endogenous crustin data indicate these peptides are necessary effectors in the shrimp immune response.