Molecular Cloning and Functional Characterization of Antimicrobial Peptides Brevinin-1ULf and Ulmin-1ULa in the Skin of the Newly Classified Ryukyu Brown Frog Rana ulma

摘要

Antimicrobial peptides (AMPs) were previously isolated from the skin of the Ryukyu brown frog Rana okinavana. However, this species has recently been reclassified as two species, i.e., Rana kobai and Rana ulma. As a result, it was determined that AMPs isolated from R. okinavana were in fact products of R. kobai, but not of R. ulma. In the present study, we collected skin samples from the species R. ulma and cloned twelve cDNAs encoding AMP precursors for the acyclic brevinin-1ULa--1ULf, the temporin-ULa-ULc, ranatuerin-2ULa, japonicin-1ULa, and a novel peptide using reverse-transcription polymerase chain reaction techniques. The deduced amino acid sequence of the novel peptide had a high similarity to those of Rana chensinensis chensinin-1CEa-1CEc, which were cloned by Zhao et al. (2011), but had a low similarity with R. chensinensis chensinin-1, which was cloned by Shang et al. (2009). To avoid confusion with these two different chensinin-1 families, we termed our peptide ulmin-1. Among these peptides, we focused on two peptides, brevinin-1ULf and ulmin-1ULa, and examined the antimicrobial and cytotoxic activity of their synthetic replicates. In broth microdilution assays, growth inhibitory activities against Staphylococcus aureus, Bacillus cereus, and Candida albicans were detected for brevinin-1ULf but not for ulmin-1ULa, whereas scanning electron microscopic observations revealed that both peptides induce morphological abnormalities in these microbes. In addition, binding activity of ulmin-1ULa to the bacterial cell wall component lipoteichoic acid was higher than that of brevinin-1ULf. In contrast, hemolytic and cytotoxic activities of brevinin-1ULf were stronger than those of ulmin-1ULa.