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

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., ,, ,, ) 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. 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.

Nisin J, a Novel Natural Nisin Variant, Is Produced by Staphylococcus capitis Sourced from the Human Skin Microbiota

“…The Gly18Thr change is also interesting, as it is observed in nisins H, U, U 2 , P, and O 1-3 and is proposed to be modified to dehydrobutyrine (Dhb), in light of the dehydration observed in a M17Q-G18T derivative of nisin Z (29). At position 20, nisin J has a highly hydrophobic residue, phenylalanine, compared to the polar asparagine in nisin A. Li et al (30) found that extending the C terminus of nisin improves both its ability to permeate membranes and its inhibitory potential against Gram-negative bacteria. Therefore, nisin J's longer C terminus (compared to other nisin variants) could be more attracted to negatively charged cell membranes resulting in enhanced membrane insertion, which may be responsible for its broader host range.…”

Bioengineering nisin to overcome the nisin resistance protein

“…Bioengineering (engineering within the cell) and the use of synthetic biology‐based ( in vitro engineering) approaches have been important for advancing our understanding of the fundamentals of bacteriocin activity and structure–function relationships and also in designing novel peptides with enhanced functionalities (activity and/or stability) which make them more attractive from a clinical perspective (Cotter et al , ). Several examples of bioengineered variants of the prototypical lantibiotic nisin have been generated that provide examples of how lantibiotic functionality can be improved through single, or a small number of residue replacements (Kuipers et al , ; Healy et al , ; Ge et al , ; Zhou et al , ; Li et al , ).…”

“…This polycyclic, 34 amino acid peptide (Fig. ) exhibits antibacterial activity against a wide range of Gram‐positive bacteria and a small number of Gram‐negative bacteria including Escherichia coli, Helicobacter pylori and Acinetobacter baumannii (Kim et al , , Kuwano et al , , Li et al ., ). Nisin exerts its antimicrobial activity both by pore formation and by inhibition of cell wall synthesis through specific binding to lipid II, an essential precursor of the bacterial cell wall (Breukink et al , ; Wiedemann et al , ; Hsu et al , ).…”

Nisin reduces uterine inflammation in rats by modulating concentrations of pro‐ and anti‐inflammatory cytokines

“…Nisin, produced by Lactococcus lactis , has been shown to display very potent activity against Staphylococcus aureus ( S aureus ) and is the most extensively studied antibiotic and is used in the treatment of numerous diseases . Mare's milk is a popular traditional fermented milk from the Mongolian plateau that uses.…”