Regulation of the Rana sylvatica brevinin-1SY antimicrobial peptide during development and in dorsal and ventral skin in response to freezing, anoxia, and dehydration

Abstract

Brevinin-1SY is the only described antimicrobial peptide (AMP) of Rana sylvatica. As AMPs are important innate immune molecules that inhibit microbes, this study examined brevinin-1SY regulation during development and in adult frogs in response to environmental stress. The brevinin-1SY nucleotide sequence was identified and used for protein modeling. Brevinin-1SY was predicted to be an amphipathic, hydrophobic, alpha helical peptide that inserts into a lipid bilayer. Brevinin-1SY transcripts were detected in tadpoles and were significantly increased during the later stages of development. Effects of environmental stress (24 h anoxia, 40% dehydration or 24 h frozen) on the mRNA levels of brevinin-1SY in the dorsal and ventral skin were examined. The brevinin-1SY mRNA levels were increased in dorsal and ventral skin of dehydrated frogs, and in ventral skin of anoxic frogs, compared with controls (non-stressed). Brevinin-1SY protein levels in peptide extracts of dorsal skin showed a similar, but not significant, trend to that of brevinin-1SY mRNA levels. Antimicrobial activity of skin extracts from control and stressed animals were assessed for Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Botrytis cinerea, Rhizopus stolonifer and Pythium sulcatum using disk diffusion assays. Peptide extracts of dorsal skin from anoxic, frozen and dehydrated animals showed significantly higher inhibition of E. coli and P. sulcatum than from control animals. In ventral skin peptide extracts, significant growth inhibition was observed in frozen animals for E. coli and P. sulcatum, and in anoxic animals for B. cinerea, compared with controls. Environmental regulation of brevinin-1SY may have important implications for defense against pathogens.