FRAGMENTS OF BETA-THYMOSIN FROM THE SEA-URCHIN PARACENTROTUS LIVIDUS AS NOVEL ANTIMICROBIAL PEPTIDES AGAINST STAPHYLOCOCCAL BIOFILMS

With the aim to face the threat of pathogen biofilms intrinsically resistant to conventional antibiotics, we focused on coelomocytes, the immune mediators in echinoderms, as source of novel antimicrobial peptides (AMPs). The proteic fraction <5kDa from coelomocytes cytosol of the sea-urchin Paracentrotus lividus (5-CC) was tested against a group of Gram positive and Gram negative pathogen reference strains. The 5-CC of P. lividus resulted active against all tested strains at concentrations ranging from 15.8 to 253.7 mg/mL. The ability to prevent staphylococcal biofilm formation was evaluated against the biofilm of clinical strain S.epidermidis 1457 using live/dead staining in combination with confocal laser scanning microscopy. In order to detect and determine the sequence of antimicrobial peptides, the 5-CC was subjected to RP-HPLC/nESI-MSMS. We demonstrated the presence of three small peptides belonging to the sequence segment 9–41 of a beta-thymosin of P. lividus (NCBInr acc. no gi|22474470) whose molecular mass is 4592 Da. In particular, the smallest peptide, the fragment 9-19 with a molecular mass of 1251.7 Da, presents the chemical-physical characteristics of AMPs: a net positive charge due to the excess of lysine residues; the ability to form alpha-helices and the presence of at least three hydrophobic residues on the same surface. Hydrophobic and charged residues may permit interactions with bacterial membranes. Moreover, it showed a similarity with already described AMPs produced by a variety of organisms, for instance with Jelleine-III produced by Apis mellifera. Such novel AMPs from beta-thymosin have a high potential as anti-biofilm agents, because they can also act on slow-growing or even non-growing bacteria that exhibit a reduced susceptibility to conventional antibiotics and represent a reservoir for recurrent biofilm associated infections