title: Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci.
abstract: We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide.
authors.affiliation: Department of Medical Microbiology, School of Medicine, University of Manchester, Oxford Road, Manchester, United Kingdom.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Sandiford
authors.forename: Mathew
authors.initials: M
authors.lastname: Upton
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D001430:Bacteriocins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D055808:Drug Discovery; D016983:Enterococcus / Q000187:drug effects* / Q000254:growth & development; D004926:Escherichia coli; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013210:Staphylococcus / Q000187:drug effects* / Q000254:growth & development; D013212:Staphylococcus epidermidis / Q000235:genetics / Q000378:metabolism*; D020713:Vancomycin Resistance
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001430:Bacteriocins; D011994:Recombinant Proteins; C574453:epidermicin NI01, Staphylococcus epidermidis