Systematic characterization of position one variants within the lantibiotic nisin

Abstract

Lantibiotics are a growing class of natural compounds, which possess antimicrobial activity against a broad range of Gram-positive bacteria. Their high potency against human pathogenic strains such as MRSA and VRE makes them excellent candidates as substitutes for classic antibiotics in times of increasing multidrug resistance of bacterial strains. New lantibiotics are detected in genomes and can be heterologously expressed. The functionality of these novel lantibiotics requires a systematic purification and characterization to benchmark them against for example the well-known lantibiotic nisin. Here, we used a standardized workflow to characterize lantibiotics consisting of six individual steps. The expression and secretion of the lantibiotic was performed employing the promiscuous nisin modification machinery. We mutated the first amino acid of nisin into all proteinaceous amino acids and compared their bactericidal potency against sensitive strains as well as strains expressing nisin resistance proteins. Interestingly, we can highlight four distinct groups based on the residual activity of nisin against sensitive as well as resistant L. lactis strains.

Figure 1

Scheme of lantibiotic characterization (a) the lantibiotic nisin and its I₁ mutants (b). The characterization of novel lantibiotics or variants is based on a six steps protocol (a). I-II are cloning and expression steps (yellow box). Step III covers peptide purification (red box). Step IV represents the activation of the peptide (orange box). Step V is the MS-analysis (green box), while step VI represents the antimicrobial activity of the lantibiotic (blue box). The lantibiotic nisin (NisA) can be dissected into an N-terminal region (with lanthionine ring A and the methyl-lanthionine rings B and C), a hinge region and a C-terminal region (with the intertwined methyl-lanthionine rings D and E) (b). The dehydrated amino acids dehydroalanine (dha) and dehydrobutyrine (dhb) (former serines and threonines) are highlighted in yellow. The coupled cysteine residues to dehydrated amino acids are highlighted in orange. The thioether bonds between the (methyl-) lanthionine are marked with a red S. The position one isoleucine is highlighted in red and exchanged to X amino acid from the four different groups of natural amino acids.

Figure 2

Growth inhibition assay of strain NZ9000-Cm in the presence of nisin and the corresponding I₁ variants. The lantibiotic nisin A (WT) and its I₁ mutants were used for growth inhibition (IC₅₀) against strain NZ9000-Cm. IC₅₀ values were grouped in four different sub-groups (group 1: aa M-G, green; group 2: aa W-Y, yellow; group 3: aa C-S, magenta; group 4: aa K-D, orange), Values represents the average of at least five independent measurements and the errors report the standard deviation of the mean (SDM). The nisin variant I₁P was not cleaved by NisP (*) and no growth inhibition assay was conducted. The nisin variants within the forth group marked with (**) showed less antimicrobial activity (IC₅₀ > 500 nM).

Figure 3

Growth inhibition assay of strains NZ9000-NisI, NZ9000-NisFEG, NZ9000-SaNSR and NZ9000-SaNsrFP in the presence of nisin and the corresponding I₁ variants. The lantibiotic nisin A (WT) and its I₁ mutants were used for growth inhibition (IC₅₀) with strains NZ9000-NisI, NZ9000-NisFEG, NZ9000-SaNSR and NZ9000-SaNsrFP. The IC₅₀ values were grouped in sub-groups of the I₁ mutants. The group 1 with the aa M-G (a), group 2 with the aa W-Y (b), group 3 with the aa C-S (c) and group 4 with the aa K-H (d). Values represent the average of at least five independent measurements and the errors report the standard deviation of the mean (SDM).

Figure 4

Fold of resistance of strains NZ9000-NisI, NZ9000-NisFEG, NZ9000-SaNSR and NZ9000-SaNsrFP in comparison to NZ9000-Cm. The resistance of the strains NZ9000-NisI, NZ9000-NisFEG, NZ9000-SaNSR and NZ9000-SaNsrFP towards the lantibiotic nisin A (WT) and its I₁ mutants are presented as fold of resistance. The fold of resistance values were grouped in sub-groups of the I₁ mutants. The group 1 with the aa M-G (a), group 2 with the aa W-Y (b), group 3 with the aa C-S (c) and group 4 with the aa K-H (d). Values represent the average of at least five independent measurements and the errors report the standard deviation of the mean (SDM).