Abstract
Bactofencin A is a novel class IId bacteriocin, produced by the intestinal isolate Lactobacillus salivarius DPC6502, which has potent activity against medically significant pathogens including Staphylococcus aureus. This bacteriocin is unusual in that it has a highly cationic N terminus and a single disulfide bond between Cys7 and Cys22, resulting in a large C terminal loop. In this study, a library of synthetic bactofencin A variants were screened against the mastitis isolate, S. aureus DPC5246, to identify key residues responsible for activity. It was apparent that substituting either cysteine of the disulfide bond with either serine or alanine significantly reduced the activity of the bacteriocin, confirming the importance of the C terminal loop. Substituting N terminal amino acids with alanine had no effect on activity, whereas sequential removal of the N terminal positively charged residues resulted in an increasingly inactive peptide. A complete (synthetic) alanine scanning analysis revealed that the residues between Val9 and Gly17 were most affected by substitution suggesting that this area has a major influence on the potency of the bacteriocin. Substituting residues in the loop region between Cys7 and Cys22 for D-amino acid equivalents had a more detrimental effect on activity than L-alanine substitutions. Specifically Y10A, N11A, P15A and T16A are active at 4, 16, 1 and 16 μM respectively while their D equivalents were inactive at 1000 μM, the highest concentration tested. Ultimately, this study identifies the critical features in the primary structure of the bacteriocin which gives it such potent activity against pathogenic staphylococci.
Highlights
Concerns about the increased incidence of antimicrobial resistance (AMR) against human pathogens have led to calls for global efforts to combat this worrying phenomenon
Synthetic peptides are initially synthesized without a disulfide bond, this bond appears to form naturally given that we can detect it by MALDI TOF MS
Synthetic peptides were HPLC purified, resuspended in sodium phosphate buffer until disulfide bond formation occurred, as confirmed by MALDI TOF MS, and HPLC purified for a second time to obtain pure peptide with an intact disulfide bond
Summary
Concerns about the increased incidence of antimicrobial resistance (AMR) against human pathogens have led to calls for global efforts to combat this worrying phenomenon. The Global Antimicrobial Resistance Surveillance System (GLASS) was set up in 2015 to standardise the collection and sharing of data on AMR at a global level and to promote coordinated action In support of this initiative the WHO recently surveyed the development of new antibiotics in the clinical pipeline against priority pathogens and found that there is particular need for new classes of antimicrobial to abate the threat of AMR. The rational design of novel antimicrobials is rapidly evolving via the use of bioengineering to generate novel bacteriocin variants with enhanced functionality This has been realised through the recent generation of both one and two peptide bacteriocins with greater activity against food-borne and medically significant Gram-positive and Gram-negative pathogens[12,13,14,15,16,17]. The identification of enhanced derivatives has been realised following initial studies in which saturation or scanning mutagenesis have been employed to reveal key important residues and structures within the peptide[12]
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