clavanin-a and Hemolysis

Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent.

Topics: Animals; Antimicrobial Cationic Peptides; Antiprotozoal Agents; Arthropod Proteins; Blood Proteins; DNA-Binding Proteins; Fetal Blood; Hemolysis; Horseshoe Crabs; Humans; Invertebrate Hormones; Leishmania braziliensis; Magainins; Mytilus edulis; Penaeidae; Peptides; Peptides, Cyclic; Trypanocidal Agents; Trypanosoma cruzi; Urochordata; Xenopus laevis

We cloned the precursor of a novel peptide from a cDNA library prepared from pharyngeal tissues of the tunicate, Styela clava. Its sequence predicted a histidine-rich, amidated 23-residue peptide (FLRF(IG)SVIHGIGHLVHHIGVAL-NH2) that we named clavaspirin. A synthetic clavaspirin was prepared and it was found that it killed Gram-positive and Gram-negative bacteria, permeabilized the outer and inner membranes of Escherichia coli, lysed phosphatidylglycerol (POPG) liposomes, and was potently haemolytic towards human and bovine erythrocytes. Each of these activities was performed more effectively at an acidic pH. Circular dichroism measurements of synthetic clavaspirin revealed a largely alpha-helical structure and polarized and residue-specific FTIR spectrometry showed that its association with phospholipid membranes was influenced by pH. Peptides such as clavaspirin may equip tunicate haemocytes to mediate cytotoxicity and participate in antimicrobial defence.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Bacteria; Base Sequence; Blood Proteins; Blotting, Northern; Cell Membrane Permeability; Circular Dichroism; Cloning, Molecular; Hemolysis; Humans; Liposomes; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Sequence Homology, Amino Acid; Spectroscopy, Fourier Transform Infrared; Urochordata