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Nano-engineering the Antimicrobial Spectrum of Lantibiotics: Activity of Nisin against Gram Negative Bacteria.

ABSTRACT: Lantibiotics, bacteria-sourced antimicrobial peptides, are very good candidates for effective and safe food additives. Among them, nisin is already approved by the EU and FDA, and has been used in food preservation for the past 40 years. Now, there is a possibility and strong interest to extend its applicability to biomedicine for designing innovative alternatives to antibiotics. The main obstacle is, however, its naturally narrow spectrum of antimicrobial activity, focused on Gram positive bacteria. Here we demonstrate broadening nisin's spectrum to Gram negative bacteria using a nano-engineering approach. After binding nisin molecules to the surface of gold nano-features, uniformly deposited on spherical carbon templates, we created a nanocomposite with a high density of positively charged groups. Before assembly, none of the components of the nanocomposite showed any activity against bacterial growth, which was changed after assembly in the form of the nanocomposite. For the first time we showed that this type of structure enables interactions capable of disintegrating the wall of Gram negative bacteria. As confirmed by the nisin model, the developed approach opens up new horizons for the use of lantibiotics in designing post-antibiotic drugs.

SUBMITTER: Vukomanovic M

PROVIDER: S-EPMC5489483 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Nano-engineering the Antimicrobial Spectrum of Lantibiotics: Activity of Nisin against Gram Negative Bacteria.

Vukomanović Marija M Žunič Vojka V Kunej Špela Š Jančar Boštjan B Jeverica Samo S Podlipec Rok R Suvorov Danilo D

Scientific reports 20170628 1

Lantibiotics, bacteria-sourced antimicrobial peptides, are very good candidates for effective and safe food additives. Among them, nisin is already approved by the EU and FDA, and has been used in food preservation for the past 40 years. Now, there is a possibility and strong interest to extend its applicability to biomedicine for designing innovative alternatives to antibiotics. The main obstacle is, however, its naturally narrow spectrum of antimicrobial activity, focused on Gram positive bact ...[more]

PMID: 28659619

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Project description:Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial compounds containing lanthionine and methyl-lanthionine residues. Nisin, one of the most extensively studied and used lantibiotics, has been shown to display very potent activity against Gram-positive bacteria, and stable resistance is rarely observed. By binding to lipid II and forming pores in the membrane, nisin can cause the efflux of cellular constituents and inhibit cell wall biosynthesis. However, the activity of nisin against Gram-negative bacteria is much lower than that against Gram-positive bacteria, mainly because lipid II is located at the inner membrane, and the rather impermeable outer membrane in Gram-negative bacteria prevents nisin from reaching lipid II. Thus, if the outer membrane-traversing efficiency of nisin could be increased, the activity against Gram-negative bacteria could, in principle, be enhanced. In this work, several relatively short peptides with activity against Gram-negative bacteria were selected from literature data to be fused as tails to the C terminus of either full or truncated nisin species. Among these, we found that one of three tails (tail 2 [T2; DKYLPRPRPV], T6 [NGVQPKY], and T8 [KIAKVALKAL]) attached to a part of nisin displayed improved activity against Gram-negative microorganisms. Next, we rationally designed and reengineered the most promising fusion peptides. Several mutants whose activity significantly outperformed that of nisin against Gram-negative pathogens were obtained. The activity of the tail 16 mutant 2 (T16m2) construct against several important Gram-negative pathogens (i.e., Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes) was increased 4- to 12-fold compared to that of nisin. This study indicates that the rational design of nisin can selectively and significantly improve its outer membrane-permeating capacity as well as its activity against Gram-negative pathogens.IMPORTANCE Lantibiotics are antimicrobial peptides that are highly active against Gram-positive bacteria but that have relatively poor activity against most Gram-negative bacteria. Here, we modified the model lantibiotic nisin by fusing parts of it to antimicrobial peptides with known activity against Gram-negative bacteria. The appropriate selection of peptidic moieties that could be attached to (parts of) nisin could lead to a significant increase in its inhibitory activity against Gram-negative bacteria. Using this strategy, hybrids that outperformed nisin by displaying 4- to 12-fold higher levels of activity against relevant Gram-negative bacterial species were produced. This study shows the power of modified peptide engineering to alter target specificity in a desired direction.

Project description:Antimicrobial peptides (AMPs) are ubiquitous amongst living organisms and are part of the innate immune system with the ability to kill pathogens directly or indirectly by modulating the immune system. AMPs have potential as a novel therapeutic against bacteria due to their quick-acting mechanism of action that prevents bacteria from developing resistance. Additionally, there is a dire need for therapeutics with activity specifically against Gram-negative bacterial infections that are intrinsically difficult to treat, with or without acquired drug resistance. Development of new antibiotics has slowed in recent years and novel therapeutics (like AMPs) with a focus against Gram-negative bacteria are needed. We designed eight novel AMPs, termed PHNX peptides, using ab initio computational design (database filtering technology combined with the novel positional analysis on APD3 dataset of AMPs with activity against Gram-negative bacteria) and assessed their theoretical function using published machine learning algorithms, and finally, validated their activity in our laboratory. These AMPs were tested to establish their minimum inhibitory concentration (MIC) and half-maximal effective concentration (EC50) under CLSI methodology against antibiotic resistant and antibiotic susceptible Escherichia coli and Staphylococcus aureus. Laboratory-based experimental results were compared to computationally predicted activities for each of the peptides to ascertain the accuracy of the computational tools used. PHNX-1 demonstrated antibacterial activity (under high and low-salt conditions) against antibiotic resistant and susceptible strains of Gram-positive and Gram-negative bacteria and PHNX-4 to -8 demonstrated low-salt antibacterial activity only. The AMPs were then evaluated for cytotoxicity using hemolysis against human red blood cells and demonstrated some hemolysis which needs to be further evaluated. In this study, we successfully developed a design methodology to create synthetic AMPs with a narrow spectrum of activity where the PHNX AMPs demonstrated higher antibacterial activity against Gram-negative bacteria compared to Gram-positive bacteria. Thus, these peptides present novel synthetic peptides with a potential for therapeutic use. Based on our findings, we propose upfront selection of the peptide dataset for analysis, an additional step of positional analysis to add to the ab initio database filtering technology (DFT) method, and we present laboratory data on the novel, synthetically designed AMPs to validate the results of the computational approach. We aim to conduct future in vivo studies which could establish these AMPs for clinical use.

Dataset Information

Nano-engineering the Antimicrobial Spectrum of Lantibiotics: Activity of Nisin against Gram Negative Bacteria.

Publications

Nano-engineering the Antimicrobial Spectrum of Lantibiotics: Activity of Nisin against Gram Negative Bacteria.

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