Bacteriocins are ribosomally synthesized antimicrobial peptides. They are naturally secreted by bacteria in a mechanism to selectively kill other bacteria in the competing environment. Numerous bacteriocins diverse in structure and mode of action have been identified, however, they commonly demonstrate bactericidal activity through pore formation or general destruction of the cell membrane.
Aureocin A53, Epidermicin NI01, Lacticin Z, and Lacticin Q are unmodified, type IId bacteriocins that have each individually demonstrated bactericidal activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. They are homologs with high sequence similarity and are characterized as highly cationic and containing several hydrophobic side chains. The positive portions of the peptides are believed to initiate interaction with the negatively charged lipid bilayer, followed by the hydrophobic functional groups integrating within the bilayer causing pore formation or in some cases, general membrane destruction of target cells. By linking two different bacteriocins with a three glycine residue that connects the C-terminal of one peptide to the N-terminal of the other, we developed hybrid bacteriocins with enhanced bactericidal effects on methicillin-resistant Staphylococcus aureus.
Our proposed study involves creating hybrid combinations of the bacteriocins Lacticin Z, Lacticin Q, Aureocin A53, and Epidermicin NI01 by connecting two of them with three glycine residues. These bacteriocins are homologous to one another and have shown effective inhibitory activity against MRSA strains. The peptides are highly cationic and contain hydrophobic side chains, which enables the bacteriocins to bind and penetrate the negatively charged phospholipid bilayer of the pathogenic strain (Netz, 2002). The study involves transformation of our plasmids into Escherichia coli, IPTG induced protein expression, cell lysis, and protein purification. After extraction of our purified proteins, we plan to observe if the hybrid bacteriocins can have a synergistic effect and compare their cytotoxicity over time to that of the individual bacteriocins against Methicillin-resistant Staphylococcus aureus. This will be done by conducting two assays: MIC (minimum inhibitory concentration) and the spot-on-lawn assay. If these hybrids show a lower MIC and greater cytotoxicity than those of the individual bacteriocins, and a mixture of the single bacteriocins, this technique can be applied to optimize the activity of bacteriocins. This can not only be applied to bacteriocins that are presently used in food preservation, but it also advances the possibility of using bacteriocins as therapeutic agents for bacterial infections, particularly those with antibiotic resistance.
1)Netz DJA, Bastos MCF, Sahl HG. Mode of action of the antimicrobial peptide aureocin A53 from Staphylococcus aureus. Appl Environ Microbiol. 2002;68(11):5274–5280. Doi: 10.1128/AEM.68.11.5274-5280.2002.
2)Sandiford S, Upton M. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against staphylococci. Antimicrob Agents Chemother. 2012;56:1539–1547. doi: 10.1128/AAC.05397-11.