Characterization of Crustin, a Putative Antimicrobial Peptide from Litopenaeus vannamei

Jessica Shockey¹, Nuala O’Leary^1,2, and Paul S. Gross^1,2

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC

As invertebrates, shrimp rely solely upon an innate, non-adaptive immune system to survive the multiple stresses encountered in the marine environment.  Both in aquaculture and the wild, these animals are subject to bacterial, fungal, and viral pathogens.  Little is known about shrimp immunity, especially at the molecular level.  However, invertebrate immune response is known to include both cellular responses (e.g. phagocytosis) and humoral responses (e.g. secretion of antimicrobial peptides).  Antimicrobial peptides (AMPs) are important in defense against pathogens in most organisms.  Preliminary work on an AMP found in shrimp, the crustins, shows it to be active against gram positive bacteria.  Sequence homology also indicates a possible C-terminal protease inhibitor domain that could be acting in conjunction with or independent from the bactericidal activity.  In order to study this protein further, a recombinant of the carboxy-terminal unique domain (CTU) of crustin containing both the AMP and protease inhibitor domains was produced in a bacterial expression system, purified, and a polyclonal antibody has been prepared.  Utilizing this antibody, the crustin family of AMPs has been partially characterized.  The native peptide was isolated directly from the animal and examined extensively for its antimicrobial and protease inhibitor activity.  In addition, expression profiling after immune challenge by western blot was used to determine patterns of regulation.