| Abstrakt: |
Because tunicates rely on innate immunity, their hemocytes are important contributors to host defense. Styela clava, a solitary ascidian, have eight hemocyte subtypes. Extracts of their total hemocyte population contained multiple small (2-4 kDa) antimicrobial peptides. When purified, these fell into two distinct families that were named styelins and clavanins.Styelins A-E are phenylalanine-rich, 32 residue peptides with activity against marine bacteria and human pathogens. They show considerable sequence homology to pleurocidins, antimicrobial peptides of the flounder, Pseudopleuronectes americanus. Styelin D, one of the five styelins identified by peptide isolation and cDNA cloning, was remarkable in containing 12 post-translationally modified residues, including a 6-bromotryptophan, two monohydroxylysines, four 3,4-dihydroxyphenylalanines (DOPA), four dihydroxylysines and one dihydroxyarginine. These modifications enhanced Styelin D's bactericidal ability at acidic pH and high salinity. A novel histochemical stain for DOPA suggested that Styelin D was restricted to granulocytes.Clavanins A-E are histidine-rich, 23 residue peptides that are C-terminally amidated and most effective at acidic pH. Clavaspirin is a newly described family member that also has potent cytotoxic properties. By immunocytochemistry, clavanins were identified in the granules of five eosinophilic granulocyte subtypes and in macrophage cytoplasm.Transmission and scanning electron micrographs of methicillin-resistant Staphylococcus aureus (MRSA) and E. coli that had been treated with Styelin D and clavaspirin suggested that both peptides induced osmotic disregulation. Treated bacteria manifested cytoplasmic swelling and extrusion of cytoplasmic contents through their peptidoglycan cell wall. The diverse array of antimicrobial peptides in S. clava hemocytes constitutes an effective host defense mechanism. |
