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, plasmidencoded 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.T he continued emergence of infections caused by multiresistant bacteria reinforces the urgent requirement for the identification and development of novel antibiotics. Bacteriocins are a group of agents that can show good activity against a range of human pathogens, including viruses, and have the potential to aid the fight against multiresistant bacteria (14,33,43).Bacteriocins are ribosomally synthesized antimicrobial peptides produced to facilitate niche competition (26, 28). The inhibitory spectrum of these agents is predominantly directed toward bacteria that are closely related to the producer strain (22, 41). Due to their novel modes of action and activity against a range of multiresistant bacteria, these inhibitors could potentially be extremely useful in the treatment of multiple infections and have already been extensively investigated for their impact on prevention of food spoilage (41).Bacteriocins exhibit a relatively narrow range of inhibition compared to the broad spectrum of classical antimicrobial agents; this, however, is thought to be a potential advantage regarding resistance development as a narrow-spectrum agent...

Identification of the genes involved in the secretion and self-immunity of lacticin Q, an unmodified leaderless bacteriocin from Lactococcus lactis QU 5

“…LnqQ/Z have no significant similarity to any members of the enterocin L50 family; in contrast, they share some similarities (46~48 %) to other leaderless bacteriocins, such as BHT-B from Streptococcus rattus strain BHT (Hyink et al, 2005), aureocin A53 from Staphylococcus aureus strain A53 (Netz et al, 2002) and epidermicin NI01 from Staphylococcus epidermidis strain 224 (Sandiford & Upton, 2012). Aureocin A53 and epidermicin NI01 are both plasmid encoded and share similar genetic organizations of their gene loci, suggesting the involvement of those genes (seven genes, not including the structural gene) in the secretion and/or the immunity of these two bacteriocins (Nascimento et al, 2012;Sandiford & Upton, 2012). On the other hand, the gene locus of BHT-B shows a different organization from the two bacteriocin loci mentioned above, thus indicating a variation of this type of bacteriocin gene locus.…”

Bifunctional Gene Cluster lnqBCDEF Mediates Bacteriocin Production and Immunity with Differential Genetic Requirements

“…The other leaderless bacteriocins genetically characterized to date have only one ABC-type transporter encoded in their gene loci (5,(18)(19)(20)(21)(22)(23). This may indicate that leaderless bacteriocins have in common the feature of having one dedicated ABC transporter mediate both secretion and immunity.…”

A novel thermotolerant and acidotolerant peptide produced by a Bacillus strain newly isolated from a fermented food (kimchi) shows activity against multidrug-resistant bacteria

“…BPC61 remained completely stable at pH 2.0-10.0 and up to 80 • C. Its activity decreased sharply at or above 100 • C. Stability of BCP61 against pH and temperature is comparable with epidermicin NI01 [14] and bacteriocin produced by Bacillus thuringiensis strain B439 [4].…”

“…Importantly, BCP61 displayed an antagonistic effect against multidrug-resistant (MDR) pathogens such as MRSA, VRSA and VRE, which was stronger than that shown by bacitracin and vancomycin. There are other reports of AMPs effective against MDR and non-MDR pathogens of Bacillus [7,8,14]. Although direct comparison is unavailable, the anti-MRSA activity of BCP61 appears much stronger than that of two peptide antibiotics from B. licheniformis DSM displaying a low range of inhibitory zone [8] against most of the tested MRSA strains.…”

“…Although direct comparison is unavailable, the anti-MRSA activity of BCP61 appears much stronger than that of two peptide antibiotics from B. licheniformis DSM displaying a low range of inhibitory zone [8] against most of the tested MRSA strains. The antimicrobial activity of BCP61 against MRSA and VRE strains is relatively weaker than that of epidermicin NI01 which showed a nanomolar range of MIC values against the strains [14].…”