Cloning of a crustin-like, single whey-acidic-domain, antibacterial peptide from the haemocytes of the European lobster, Homarus gammarus, and its response to infection with bacteria

Degenerate PCR was used to isolate a 221-base pair nucleotide sequence of a new crustin-like antibacterial peptide from the haemocytes of the European lobster, Homarus gammarus. Rapid amplification of cDNA ends was used to extend the sequence to determine the complete open reading frame and un-translated regions. The inferred amino acid sequence of this peptide was found to be similar to crustin-like peptides isolated for several species of shrimp as well as the shore crab, Carcinus maenas. The sequence also contains a single-whey-acidic protein (WAP) domain, similar to novel antibacterial single-whey-acidic domain (SWD) peptides that have been recently described in the tiger shrimp, Penaeus monodon, and the Pacific white shrimp, Litopenaeus vannamei. Real-time PCR was used to analyse the expression of the gene coding for this peptide. The gene is up regulated after inoculation with the Gram-positive lobster pathogen Aerococcus viridans var. homari but down regulated after inoculation with the Gram-negative bacteria Listonella anguillarum. Phylogenetic analysis of this new peptide shows that it is most related to other antimicrobial crustin peptides and that the crustins are only distantly related to the antibacterial SWD peptides recently described.