A combined mass spectrometric and cDNA sequencing approach to the isolation and characterization of novel antimicrobial peptides from the skin secretions of Phyllomedusa hypochondrialis azurea.
Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent anti-microbial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea. Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. 3'-RACE reactions used primers designed for the highly conserved 5'-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 microM when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli, Staphylococcus aureus and Micrococcus luteus.
Thompson
Alan Hunter
AH
Institute of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co Londonderry BT52 1SA, Northern Ireland, UK.
Bjourson
Anthony John
AJ
Orr
David Francis
DF
Shaw
Chris
C
McClean
Stephen
S
D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001001:Anura / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000379:methods; D012867:Skin / Q000378:metabolism*
D029845:Amphibian Proteins; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; C070905:dermaseptin