Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio

The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1-3 (a novel lymphocyte antigen 6 (Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1-3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1-3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. We also showed that recombinant proteins Ly2.1-3 (rLy2.1-3) were able to bind to the Gram-negative bacteria A. hydrophila, E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1-3 could specifically bind to LPS. Finally, we found that Ly2.1-3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1-3 can function as AMPs.