Antibacterial and detoxifying activity of NZ17074 analogues with multi-layers of selective antimicrobial actions against Escherichia coli and Salmonella enteritidis

Na Yang,Da Teng,Jianhua Wang,Ruoyu Mao,Zhanzhan Li,Xuehui Liu,Ya Hao,Xiao Wang,Xiumin Wang

doi:10.1038/s41598-017-03664-2

Abstract

NZ17074 (N1), an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is cytotoxic. To reduce its cytotoxicity, seven N1 analogues with different structures were designed by changing their disulfide bonds, hydrophobicity, or charge. The “rocket” analogue-N2 and the “kite” analogue-N6 have potent activity and showed lower cytotoxicity in RAW264.7 cells than N1. The NMR spectra revealed that N1, N2, and N6 adopt β-sheet structures stabilized by one or two disulfide bonds. N2 and N6 permeabilized the outer/inner membranes of E. coli, but did not permeabilize the inner membranes of S. enteritidis. N2 and N6 induced E. coli and S. enteritidis cell cycle arrest in the I-phase and R-phase, respectively. In E. coli and in S. enteritidis, 18.7–43.8% of DNA/RNA/cell wall synthesis and 5.7–61.8% of DNA/RNA/protein synthesis were inhibited by the two peptides, respectively. Collapsed and filamentous E. coli cells and intact morphologies of S. enteritidis cells were observed after treatment with the two peptides. Body weight doses from 2.5–7.5 mg/kg of N2 and N6 enhanced the survival rate of peritonitis- and endotoxemia-induced mice; reduced the serum IL-6, IL-1β and TNF-α levels; and protected mice from lipopolysaccharide-induced lung injury. These data indicate that N2 and N6, through multiple selective actions, may be promising dual-function candidates as novel antimicrobial and anti-endotoxin peptides.

Highlights

Escherichia coli and Salmonella enteritidis are major food-borne pathogens that often cause outbreaks of diarrheal diseases and extraintestinal infections in both animals and humans throughout the world[1]
A series of N1 analogues were designed by changing disulfide bonds, hydrophobicity, or charge to reduce cell cytotoxicity, investigate the effects of disulfide bonds on antimicrobial activity, and develop novel cell-selective β-sheet antimicrobial peptides (AMPs) with improved antimicrobial activity
It has been demonstrated that an increase in the hydrophobic moment and net charge leads to an increase in antibacterial activity of AMPs34

Summary

Introduction

Escherichia coli and Salmonella enteritidis are major food-borne pathogens that often cause outbreaks of diarrheal diseases and extraintestinal infections in both animals and humans throughout the world[1]. To reduce cell cytotoxicity, determine the role of disulfide bonds, evaluate structure–activity relationships and improve antimicrobial activity, modification of N1 was performed based on the replacement of multiple residues by the hydrophobic/cationic residues Ala/Lys and the removal of disulfide bonds or the addition of hydrophobic Trp residue at the 1st, 3rd, 4th, 5th, 7th, 12th, 16th, and 20th positions, which generated four types of N1 variants: (i) “rocket” analogues with two disulfide bonds (Gly1,12 → Ala; Trp[4], Asn5 → Ala), (ii) a “kite” analogue with one disulfide bond (Cys3,20 → Ala), (iii) a “bullet” analogue with one disulfide bond (Cys7,16 → Ala), and iv) other linear analogues without disulfide bonds (∆Cys[3,7,16,20]; Cys3,7,16,20 → Ala) These peptides were chemically synthesized and their antimicrobial activity against gram-negative bacteria was examined. The in vivo antibacterial activity of N2 and N6 was evaluated in murine peritonitis models induced by E. coli and S. enteritidis, respectively, as well as endotoxin neutralizing activity in a murine endotoxemia model challenged with LPS

Methods

Results

Conclusion