Bioengineered nisin A derivatives with enhanced activity against both Gram positive and Gram negative pathogens

Abstract

Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.

Figure 1. Structures of natural nisin and enhanced bioengineeredvariants.

Six natural variants are known, nisin A, Z, F, Q, U and U2. Black circles indicate amino acid differences between the natural nisin variants. Broken arrows denote enhanced activity of bioengineered nisin A or Z as a result of single amino acid alterations , , , and/or combination of amino acid substitutions (joined circles) . Residues are represented in the single letter code. Post translational modifications are indicated as follows, Dha: dehydroalanine, Dhb: dehydrobutyrine, Abu: 2-aminobutyric acid, Ala-S-Ala: lanthionine, Abu-S-Ala: 3-methyllanthionine. Adapted from .

Figure 2. Bioactivity and mass spectrometry analysis of nisin A and nisin A S29G.

Growth inhibition of (A) S. aureus SA113 by the nisin A producing strains NZ9800pPTPL-nisA and NZ9800pPTPL-nisA S29G (B) Colony Mass Spectrometry analysis of nisin A (3352.63 amu) and the nisin A S29G (3322.97 amu) derivative.

Figure 3. Deferred antagonism assays of nisin A S29 derivatives against the nisin-sensitive indicator Lactococcus lactis HP.

(A) spot on lawn of producing strains on GM17 agar and overlaid with GM17 agar (0.75%) seeded with HP and (B) supernatants of producing strains in agarose-based (1%) GM17. Single letters correspond to IUPAC abbreviation code, wt = Serine.

Figure 4. Kill curve analysis of strains C. sakazakii DPC 6440, S. typhimurium UK-1 and E. coli in 33mg/L respectively of nisin A, S29A and S29G.

Figure 5. Activity of purified peptides against C. sakazakii DPC 6440.

Activity of purified peptides of nisin A, S29G and S29A (30 µM) against C. sakazakii DPC 6440 as determined by agarose gel diffusion assay using (A) peptide alone and (B) peptide in combination with polymyxin B nonapeptide (PMBN) at a concentration of 20 µg/ml. Results are expressed as total area of inhibitory zone expressed in mm². Values in bold reached statistical significance compared to the nisin control (Student’s t-test: P<0.05).