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. 2018 May 31;84(12):e00052-18.
doi: 10.1128/AEM.00052-18. Print 2018 Jun 15.

Increasing the Antimicrobial Activity of Nisin-Based Lantibiotics Against Gram-Negative Pathogens

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Free PMC article

Increasing the Antimicrobial Activity of Nisin-Based Lantibiotics Against Gram-Negative Pathogens

Qian Li et al. Appl Environ Microbiol. .
Free PMC article

Abstract

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.

Keywords: Gram-negative pathogens; antimicrobial activity; antimicrobial peptide; lantibiotic; nisin; outer membrane.

Figures

FIG 1
FIG 1
Schematic structure of prenisin with a His tag and fusions. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, β-methyllanthionine. Prenisin (gray) contains a leader peptide and a core peptide (1 to 34 amino acids). The 6 histidine residues are located at the N terminus and labeled in yellow. The ABCDE rings are marked. The structures of fusion peptides are indicated, with the linker being labeled in purple (serine and glycine), while tails with activity against Gram-negative bacteria are labeled with green. Group 1 contains full nisin and tails with activity against Gram-negative bacteria. Group 2 contains the ABCDE rings of nisin, the hinge region (serine and glycine), and tails with activity against Gram-negative bacteria.
FIG 2
FIG 2
Screening the hybrid peptides against L. lactis and E. coli CECT101. Thirty microliters of TCA-precipitated supernatants produced by L. lactis NZ9000(pIL3EryBTC/pNZ-mutant) was deposited on the wells. T1F to T11F, group 1 hybrid peptides containing full nisin fused to tails with activity against Gram-negative bacteria; T1S to T11S, group 2 hybrid peptides containing ABCDE rings of nisin and the hinge region (serine and glycine) fused to tails with activity against Gram-negative bacteria; nisA, the positive control, consisting of a TCA-precipitated supernatant of NZ900(pIL3EryBTC/pNZ-nisA); empty, negative control, consisting of the precipitated supernatant of NZ9000(pIL3EryBTC/pNZ8048). The indicator strains were L. lactis NZ9000(pNZnisP8H) (A to C) and E. coli CECT101 (D to F).
FIG 3
FIG 3
Determination of viable cells after treatment with fusion peptides. (A) Positive control for E. coli, consisting of 50 μl of a 100-fold-diluted sample from the well with E. coli alone; (B) 50 μl of a diluted sample from the well in which E. coli was treated with T16m2; (C) medium control (no bacteria were inoculated in this well); (D) positive control for A. baumannii, consisting of 50 μl of a 100-fold-diluted sample from the well with A. baumannii alone; (E) 50 μl of a 100-fold-diluted sample from the well in which A. baumannii was treated with T16m2; (F) medium control (no bacteria were inoculated in this well).

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