A New Synthetic Peptide Having Two Target of Antibacterial Action in E. coli ML35

Abstract

The increased resistance of microorganisms to the different antimicrobials available to today has highlighted the need to find new therapeutic agents, including natural and/or synthetic antimicrobial peptides (AMPs). This study has evaluated the antimicrobial activity of synthetic peptide 35409 (RYRRKKKMKKALQYIKLLKE) against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 15442 and Escherichia coli ML 35 (ATCC 43827). The results have shown that peptide 35409 inhibited the growth of these three bacterial strains, having 16-fold greater activity against E. coli and P. aeruginosa, but requiring less concentration regarding E. coli (22 μM). When analyzing this activity against E. coli compared to time taken, it was found that this peptide inhibited bacterial growth during the first 60 min and reduced CFU/mL 1 log after 120 min had elapsed. This AMP permeabilized the E. coli membrane by interaction with membrane phospholipids, mainly phosphatidylethanolamine, inhibited cell division and induced filamentation, suggesting two different targets of action within a bacterial cell. Cytotoxicity studies revealed that peptide 35409 had low hemolytic activity and was not cytotoxic for two human cell lines. We would thus propose, in the light of these findings, that the peptide 35409 sequence should provide a promising template for designing broad-spectrum AMPs.

Antimicrobial and Antibiofilm Peptides

“…In the barrel-stave model, peptides perpendicularly insert into the membrane, promoting peptide-peptide lateral interactions. In this mechanism, the AMPs' amphipathic structure plays a significant role because the hydrophilic residues generate the channels' lumen while the hydrophobic side establishes a favorable interaction with membrane lipids [41]. To date, only a few peptides, such as pardaxin and alamethicin, that act through this mechanism have been identified [42,43].…”

“…In particular, Mardirossian et al tested the antimicrobial activity of Bac5 , an N-terminal fragment of the bovine proline-rich antimicrobial peptide Bac5, on Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa, showing the inhibition of bacterial protein synthesis [40]. In addition, the synthetic peptide 35409 has been reported to inhibit cell division and induce filamentation, suggesting two different targets within a bacterial cell [41], or the lysine-peptoid hybrid, LP5, binds DNA gyrase and topoisomerase IV, causing inhibition of thee replication and ATP leakage from bacterial cells [42].…”

Roles of the Conserved Amino Acid Residues in Reduced Human Defensin 5: Cysteine and Arginine Are Indispensable for Its Antibacterial Action and LPS Neutralization

“…Because E . coli ML35 lacks lactose permease but contains a cytoplasmic β‐galactosidase, we further carried out a 2‐nitrophenyl β‐ d ‐galactopyranoside (ONPG) hydrolytic assay to monitor inner membrane permeabilization. According to the yield of o ‐nitrophenol, C/A and R/K replacements were detrimental for the disintegration of the inner membrane by HD5 RED (Figure B).…”

Shorter Antibacterial Peptide Having High Selectivity for E. coli Membranes and Low Potential for Inducing Resistance

“…A Bruker Daltonics Microflex LT mass spectrometer was used for determining the peptides’ mass/charge ratio by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS [ 30 ]. The peptide (1 mg/mL) was mixed with 1 mg/mL α-cyano-4-hydroxycinnamic acid in an 18:2.5 v/v ratio.…”

“…This peptide did not have cytotoxic activity against HeLa and HepG2 human cell lines, but did have activity against human red blood cells (hRBC) at 1.5 µM minimum haemolytic concentration (MHC). Its therapeutic index (TI) calculated for E. coli ML35 is 0.045, indicating low selectivity [ 29 ], thereby restricting its therapeutic use [ 30 ]. Considering the 35409 sequence’s antibacterial potential and the urgent need for developing new molecules having activity against microorganisms, this research was focused on obtaining short 35409-derived synthetic peptides having high selectivity for microorganisms.…”