Year of Graduation
2021
Level of Access
Open Access Thesis
Embargo Period
5-20-2021
Department or Program
Biochemistry
First Advisor
Elizabeth Stemmler
Abstract
The American lobster Homarus americanus uses its innate immune system for protection against foreign bodies and diseases. Hemocytes in the innate immune system are responsible for the rapid and effective cellular response against pathogens and infections observed in lobsters. These hemocytes, particularly semi-granulocytes and granulocytes, store antimicrobial peptides (AMPs) which specifically target and destroy microbes. Hemocyte samples from the American lobster Homarus americanus hemolymph or circulatory fluid, mixed and fractionated into separated semi-granular and granular cell samples, were analyzed for possible AMP presence. A defensin AMP, Hoa-D1, (SYVRSCSSNGGDCVYRCYGNIINGACSGSRVCCRSGGGYamide; with C representing a cysteine participating in a disulfide bond) was successfully isolated and identified by mass using reversed-phase high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Preliminary results also show the defensin AMP to be concentrated in the semi-granulocytes and granulocytes.
Hoa-D1 was isolated via HPLC fractionation. Isolated Hoa-D1 and semi-granular and granular hemocyte extracts were tested for bioactivity against the gram-negative bacteria, Escherichia coli, using the Kirby-Bauer disk diffusion assay. The assay did not show any activity, an outcome attributed to concentrations of the AMP that were too low to have any antimicrobial effect on E. coli. Subsequent work on this study should involve increasing the concentration of Hoa-D1 in test samples. Studying function of AMPs in the American lobster can provide more information on the depth of their cellular immune responses in other crustaceans, and possibly contribute to the development of novel antibiotics for treating diseases in humans.