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

Summary 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 modelling. 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 freezing) 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 frozen frogs, compared to controls (non-stressed). Brevinin-1SY protein levels in dorsal skin peptide extracts 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 the Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Botrytis cinerea, Rhizopus stolonifer, and Pythium sulcatum using disk diffusion assays. Dorsal skin peptide extracts 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 to controls. Environmental regulation of brevinin-1SY may have important implications for defense against pathogens.