152 Regulation of the antimicrobial peptide brevinin-1SY in the skin of Rana sylvatica in response to environmental stress

Abstract

Antimicrobial peptides (AMPs) are innate immune defense molecules found throughout prokaryote and eukaryote kingdoms. Brevinin-1SY is the sole AMP produced by the skin of the North American wood frog, Rana sylvatica ; a terrestrially hibernating frog that survives whole body freezing during the winter. As a consequence of freezing, R. sylvatica is also exposed to anoxia and dehydration of their cells. The objective of our study was to examine whether brevinin-1SY was regulated in the dorsal and ventral skin of R. sylvatica during 24 h anoxia, 40% dehydration, or 24 h frozen environmental stress conditions. An ethanol/trifluoroacetic acid extraction method was used to isolate the hydrophobic peptide fraction of frog skin. The antimicrobial activity of skin extracts from control (non-stressed) and stressed animals were assessed for the bacteria Escherichia coli and Bacillus subtilis , the yeast Saccharomyces cerevisiae , the molds Botrytis cinerea and Rhizopus stolonifer , and the oomycete Pythium sulcatum using disk diffusion assays. Dorsal skin peptide extracts from anoxic, frozen and, in particular, dehydrated animals showed significantly higher inhibition of E. coli and P. sulcatum than dorsal skin peptide extracts from control animals. In ventral skin peptide extracts, an increased growth inhibition was observed in frozen animals for E. coli , in anoxic animals for B. cinerea , and in frozen animals for P. sulcatum in comparison to peptide extracts from their respective controls. To assess whether the differences in antimicrobial activity were the result of changes in brevinin-1SY at the mRNA and protein level, transcript levels of brevinin-1SY were assessed in frog skin samples using semi-quantitative PCR and protein levels were analyzed by SDS-PAGE followed by silver staining and densitometry analysis. Brevinin-1SY mRNA levels were increased in dorsal and ventral skin from dehydrated frogs, and in ventral skin of frozen frogs, compared to non-stressed controls. Analysis of brevinin-1SY protein levels in the peptide extracts showed a similar trend to that of the mRNA levels in both dorsal and ventral skin. In silico analysis revealed brevinin-1SY to be a helical AMP with a hydrophobic and a hydrophilic face. Modeling of brevinin-1SY with a model membrane predicted the hydrophobic face of the helix would insert into the membrane, suggesting possible membrane disruption as the antimicrobial mechanism of action. The antimicrobial activity of brevinin-1SY in wood frog skin to select microorganisms is modulated in response to environmental stress and appears to be controlled at a transcriptional level. The ability of the wood frog to modulate AMPs during environmental stress may have important implications for frog host defense against pathogens. Source of funding: None declared. Conflict of interest: None declared. kenneth_storey@carleton.ca