Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens

Qian Li,Weihui Wu,Manuel Montalbán-López,Rubén Cebrián,Huan Ren,Oscar P Kuipers

doi:10.1038/s42003-020-01511-1

Abstract

The development and dissemination of antibiotic-resistant bacterial pathogens is a growing global threat to public health. Novel compounds and/or therapeutic strategies are required to face the challenge posed, in particular, by Gram-negative bacteria. Here we assess the combined effect of potent cell-wall synthesis inhibitors with either natural or synthetic peptides that can act on the outer-membrane. Thus, several linear peptides, either alone or combined with vancomycin or nisin, were tested against selected Gram-negative pathogens, and the best one was improved by further engineering. Finally, peptide D-11 and vancomycin displayed a potent antimicrobial activity at low μM concentrations against a panel of relevant Gram-negative pathogens. This combination was highly active in biological fluids like blood, but was non-hemolytic and non-toxic against cell lines. We conclude that vancomycin and D-11 are safe at >50-fold their MICs. Based on the results obtained, and as a proof of concept for the newly observed synergy, a Pseudomonas aeruginosa mouse infection model experiment was also performed, showing a 4 log10 reduction of the pathogen after treatment with the combination. This approach offers a potent alternative strategy to fight (drug-resistant) Gram-negative pathogens in humans and mammals.

Highlights

9 of the 12 “superbugs” listed are Gram-negative pathogens. Their outer-membrane acts as an efficient barrier to prevent various antimicrobials from reaching their targets at the inner membrane and/or the cytoplasm, which complicates the development of efficient treatments against bacteria[7,8]
Vancomycin and nisin were selected because they are potent antimicrobials against Gram-positive bacteria whereas they display low or absent activity against Gram-negative bacteria
They are considered as safe antimicrobials in food preservation[17] or in clinical application[14], and both use the same target, lipid II, to kill bacteria

Summary

Introduction

Nisin and vancomycin are potent antimicrobials inhibiting cell-wall biosynthesis by binding to lipid II at the cell membrane using two different binding motifs. They have low micromolar minimal inhibitory concentrations (MICs) but are poorly active on Gram-negative bacteria due to the presence of their outermembrane[12,13,14]. Eight, either natural or synthetic, peptides ranging in size between 11 and 23 amino acids, which exert low to modest activity against Gram-negative bacteria, were selected from literature to work as possible Gram-negative outer-membranepenetrating or -perturbing peptides, thereby exposing lipid II in the inner membrane to either nisin or vancomycin.

Methods

Results

Conclusion