Shapo, J. L.

An evaluation of antimicrobial activity in the common seawhip, Leptogorgia virgulata (Lamarck)

2006

Charleston (SC)

College of Charleston

A hallmark of the cnidarian innate immune system is the rapid, nonspecific response of antimicrobial compounds to damage, stress, or pathogen exposure. Antimicrobial activity was examined in the common seawhip, Leptogorgia virgulata (Lamarck), from South Carolina waters. Extraction and assay protocols were developed to permit identification of antimicrobial activity in crude methanol/sterile water extracts of L. virgulata. Antimicrobial activity was detected by bacterial growth inhibition assays using Escherichia coli BL21, Vibrio harveyii, Micrococcus luteus, and a Bacillus sp. isolate from a scleractinian coral (Acropora cervicornis). This research represents the first report of antimicrobial activity in L. virgulata and, of equal note, in a temperate/subtropical coral in the Atlantic Ocean. Antimicrobial activity in L. virgulata was measured at three reference and four anthropogenically impacted sites in South Carolina between May and October 2005. No obvious relationship existed between the degree of anthropogenic stress at any of the seven sites and the production of antimicrobial compounds in L. virgulata. Colonies of L. virgulata collected from the Charleston City Marina in Charleston, SC, were the only samples which did not exhibit antimicrobial activity agains t selected bacteria during the six-month study period. Antimicrobial activity was highly variable at the other six sites, differing significantly at Cherry Point Seafood Company, Patriots Point Marina, Kiawah River Bridge, Huntington Beach State Park-South Jetty, and upper 60 Bass Creek-North Inlet among points of collection throughout the study season. Reverse-phase high performance liquid chromatography (HPLC), HPLC coupled to mass spectrometry (LC-MS), and 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy were used in conjunction with a liquid growth inhibition assay system to isolate, purify, and characterize unknown metabolites from active antimicrobial fractions of L. virgulata. Corroborative MS/NMR evidence based on diagnostic molecular weights and chemical shifts validated the presence of homarine and a homarine analog in pooled fractions 14-21 and also 22-24. Homarine, a well-known emetic and antifouling metabolite, has been isolated previously from L. virgulata. In subsequent liquid growth inhibition assays, replicates of pooled fractions 14-21 inhibited the growth of V. harveyii and M. luteus, indicating that these partially purified fractions contained biologically active compounds responsible for the antimicrobial activity in L. virgulata. It was not determined whether homarine and/or a homarine analog is exclusively responsible for the antimicrobial activity in L. virgulata. The findings of this study suggest that homarine is, in part, an active constituent of the innate immune system in L. virgulata, which may act synergistically with cofactors and/or congeners in the octocoral to mount a rapid response to stressors, such as microbial invasion and disease.