Exploration of the Innate Immune System of Styela clava: Zn Binding Enhances the Antimicrobial Activity of the Tunicate Peptide Clavanin A.

Tunicates have been used as primitive models for understanding cell-mediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, β-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.