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Zoological Science
Abstract
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.
This work was supported by JSPS KAKENHI Grant Numbers JP21570068 and JP24570077 to SI, IH, TK, and SK.
The authors have no competing interests to declare.
DO, MM, MF: collection and assembly of data, data analysis and interpretation; IH, TK, SK: data analysis and interpretation, financial support, and manuscript writing; SI: conception and design, financial support, assembly data, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Supplementary material for this article is available online (URL: http://www.bioone.org/doi/suppl/10.2108/zs170084).
Supplementary Figure S1. Comparison and schematic alignment of amino acid sequences deduced from R. ulma AMP precursor homologue cDNAs with corresponding sequences of other ranid frog AMP precursors that are registered in the GenBank database.
Supplementary Figure S2. The effects of various concentrations of synthetic brevinin-1ULf in the absence (white columns) and presence (gray columns) of ulmin-1ULa on the growth of grampositive bacteria S. aureus (A) and B. cereus (B).
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