Characterization of antimicrobial activity present in the cuticle of American lobster, Homarus americanus
Mars, Margaret Anne
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American lobster is an ecologically and socioeconomically important species. In recent years the species has been affected by disease and the catch in Southern New England has fallen dramatically. In order to fully understand how and why diseases affect lobster populations, it is imperative to fully understand lobster defense mechanisms. The cuticle, previously believed to act only as a physical barrier, has recently been shown to contain antimicrobial activity. This thesis focused on characterizing this activity and attempted to identify the molecules responsible. A sterile shell extract prepared from a pool of lobster shells was active against Vibrio parahaemolyticus, Vibrio anguillarum, Escherichia coli, and Staphylococus aureus. The activity in the extract was found to be heat stabile, as activity was not decreased after boiling (100°C, 5 min.). Activity was diminished when an extract was prepared from muffled lobster shells, indicating an organic component responsible for the observed activity. Size fractioning of the extract with centrifugal filter units decreased activity in extract unable to pass through a 10kDa filter, while extract that passed through a 10kDa and 3kDa filter retained antimicrobial activity, despite having much lower protein concentrations. Fast protein liquid chromatography (FPLC) of the shell extract revealed several protein peaks. Samples corresponding to FPLC peaks had varying antimicrobial activities. In contrast, the void volume, containing inorganic and anionic material, increased bacterial growth. Tris/tricine SDS-PAGE with the FPLC fractions revealed small peptides (<7kDa) in the fractions that displayed the highest and least variable antimicrobial activity. MALDI mass spectrometry revealed peptide peaks at 1.6, 2.8, 4.6, and 5.6 kDa. A partial sequence of the 5.6kDa peptide was determined. Manipulation of the unknown amino acids in the sequence in a search with BLAST led to a non-definitive, partial match with the wasp antimicrobial peptide mastoparan. Antimicrobial peptides have been described as being cationic peptides less than 10kDa with broad-spectrum antimicrobial activity. The small size and structure of the peptides make them very stabile and able to withstand high heat. AMPs have been isolated from a wide variety of plants and animals and are an integral part of the invertebrate defense response. This thesis demonstrated that the broad-spectrum antimicrobial activity observed in the lobster shell is due to a component that is organic, cationic, heat stabile, and less than 10kDa. Based on shared characteristics with antimicrobial peptides, it is likely that the activity observed in the shell is due to an antimicrobial peptide.In addition, antimicrobial activity in the shells of a lobster population with a high incidence of Epizootic shell disease (Eastern Long Island Sound, ELIS) was compared with the activity in shells of lobsters from a population with a low incidence of the disease (Western Long Island Sound, WLIS). Extracts from the shells of WLIS lobsters had significantly higher antimicrobial activity when compared to extracts from ELIS lobsters. The antimicrobial activity in the shell may be a factor affecting susceptibility to the disease.