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.

Characterization of the nucleotide-binding domain NsrF from the BceAB-type ABC-transporter NsrFP from the human pathogen Streptococcus agalactiae

“…This variant served as a negative control in all our experiments. The lack of NTP hydrolysis for Sa NsrF H202A is in line with in vivo studies that show that this variant abolishes the activity of Sa NsrFP 8 , 40 .…”

Impact of the nisin modification machinery on the transport kinetics of NisT

“…According to these studies the observed high secretion efficiency of NisA by NisBTC was explained by a ‘channeling mechanism’ 19 , 35 . Other studies focused on the application of the nisin modification machinery to produce nisin variants or lantibiotics and secrete them by NisT 36 – 39 . Despite the in vivo analysis of NisA secretion, a first analysis with labeled precursor peptides was performed and gave first insights into the kinetics of secretion 35 .…”

Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion

“…Monitoring and quantification of the active lantibiotic by integrating the leader peptide peaks in RP-HPLC might be complicated due to alteration of running properties of the pre-lantibiotics and activated forms during the experiment and make it difficult to distinguish between similar peaks. This strategy has been used to determine the concentration of the activated lantibiotic during the production of genome-mining new lantibiotics as a hybrid containing the leader peptide of nisin (Lagedroste et al, 2019).…”

“…Later, the lipid-II binding activity of some lantibiotics was also demonstrated ( Wiedemann et al, 2001 ; Breukink and de Kruijff, 2006 ), although in some of them the mechanisms of action have yet to be clarified. Nonetheless, low cytotoxicity of lantibiotics to human cells due to the absence of their main target lipid II or negative net charge in eukaryotic membranes ( Lagedroste et al, 2019 ) along with a few examples of naturally occurring resistance ( Draper et al, 2015 ; Khosa et al, 2016a,b ; Clemens et al, 2018 ) make lantibiotics excellent lead compounds for development of new therapeutic options.…”