MENZEL, LP; TINCU, JA; SJOSTRAND, BM; LEHRER, RI: Antimicrobial peptides of tunicate hemocytes: cellular source and effects on bacterial targets.

Antimicrobial peptides are key effector molecules that equip the innate immune system to combat infections. We studied antimicrobial peptides found in the hemocytes of two tunicates, Styela clava and Styela plicata. The hemocytes of S. clava contain two families of antimicrobial peptides, Clavanins and Styelins. Clavanins are histidine-rich, C-terminally amidated peptides with 23 amino acid residues. Clavaspirin, the most recently described member of the clavanin family, also has prominent cytotoxic properties. Styelins are phenylalanine-rich, 3.7 kDa peptides with 32 residues, including (in Styelins D & E) multiple 3,4-dihydroxyphenylalanine (DOPA) residues. The hemocytes of S. plicata contain plicatamide, a DOPA-containing octapeptide with prominent antimicrobial properties. Clavanins, styelins and plicatamide increase the permeability of the outer and inner membranes of E. coli and MRSA and caused extensive potassium loss. Various morphological changes occur, including membrane alterations (ruffling, bleb formation, shedding), swelling, mesosome formation in MRSA (plicatamide only), etc. We performed immunochemical and cytochemical studies to determine which of S. clava’s hemocyte types contain clavanins and styelins. Immunogold studies identified clavanins throughout the cytoplasm of macrophages, and in the cytoplasmic granules of four different granulocyte types. A modified nitro-blue tetrazolium NBT stain that recognized DOPA-containing peptides allowed us to determine that Styelins D & E were present in approximately half of the total hemocyte population – granulocytes that also contained clavanins. Together, clavanins and styelins comprised between 10-20% of the total acid-extractable cellular protein of S. clava hemocytes.