Dual Mechanism of Bacterial Lethality for a Cationic Sequence-Random Copolymer that Mimics Host-Defense Antimicrobial Peptides

Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
Journal of Molecular Biology (Impact Factor: 4.33). 06/2008; 379(1):38-50. DOI: 10.1016/j.jmb.2008.03.047
Source: PubMed


Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.

Download full-text


Available from: Sarah E Lee
  • Source
    • "The association with the membranes may then proceed via a mechanism similar to the ''carpet'' mecha- nism[46,47]. Alternatively, the membrane disruption may be a result of aggregation of charged lipids and subsequently generation of defects between lipid domains[32]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Antibacterial polysiloxane polymers with pending tert-butylamine groups are a novel class of compounds that are compatible with silicone elastomers, but their mechanism of action is not well understood. The research into their action mechanism was conducted on a polysiloxane copolymer grafted with tert-butylaminoethyl methacrylate and covalently attached fluorescein. Fluorometric measurements results suggest that the polymer forms a stable link with bacteria. The results of β-galactosidase enzyme assay with the use of ortho-nitrophenyl-β-galactoside as a substrate show that the polymer has a damaging effect on bacterial membranes. The scanning and transmission electron micrographs of Escherichia coli cells incubated with the polymer prove further that the polymer’s site of action is bacterial cell membranes. In order to investigate the polymer interaction with bacterial membranes the fluorescein labelled polymer was incubated with bacterial cells and membranes isolation and identification method was next applied. The E. coli membrane fractions were identified by light scattering, protein content, oxidase NADH activity and N-phenylnaphtylamine fluorescence measurements, as well as electron microscopy. Oxidase NADH and N-phenylnaphtylamine were the inner membrane markers. The bacterial membranes were then tested for the presence of the polymer. The experiments gave evidence that the copolymer binds to the inner bacterial membrane. Further studies, where the copolymer was incubated with isolated mixed (inner and outer) membrane fractions, proved that the copolymer exerts more destructive effect on E. coli outer membrane. The damaging effect on the membranes is concentration dependent.
    Full-text · Article · Mar 2016 · Journal of Materials Science Materials in Medicine
  • Source
    • "E. coli ML35p, a pUC19 plasmid-transformed ML35 strain, is generally used for investigating cell membrane permeability because of its high expression levels of intracellular β-galactosidase and lack of lactose permease [30]. The cells were incubated to log-phase growth (OD600 0.4–0.6) at 37°C with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), an allolactose analog, to induce the synthesis of the pUC19-encoded β-galactosidase. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We develop a novel nanohybrid showing a strong antibacterial activity on all of the tested pathogens, including methicillin-resistant Staphylococcus auerus and silver-resistant E. coli. The nanohybrid consists of silver nanoparticles (AgNPs) supported on 1 nm-thick silicate platelets (NSPs). The AgNP/NSP nanohybrid enables to encapsulate bacteria and triggers death signals from the cell membrane. The geographic shape of the NSPs concentrates AgNPs but impedes their penetration into attached cells, mitigating the detrimental effect of silver ion deposition in applied tissues. Moreover, the tightly tethered AgNPs on NSP surface achieve a stronger biocidal effect than silver nitrate, but bypassing Ag(+) mechanism, on silver-resistant bacteria. This nanohybrid presents an effective and safe antimicrobial agent in a new perspective.
    Full-text · Article · Jun 2011 · PLoS ONE
  • Source
    • "A novel mechanism that appears to contribute to the bactericidal effects of some antimicrobial peptides is their ability to cluster anionic lipids from zwitterionic ones [15] [16] [17] [18]. Evidence presented for the formation of domains by antimicrobial agents has included DSC [16] [17] [18] [19], 31 P MAS/NMR [16] [18], FTIR [17] and 2 H NMR [20] as well as AFM in combination with polarized fluorescence microscopy [21]. In the present work, in addition to presenting evidence for anionic lipid clustering by DSC, we have directly imaged changes in membrane morphologies using freeze fracture electron microscopy. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Three Arg-rich nonapeptides, containing the same amino acid composition but different sequences, PFWRIRIRR-amide (PR-9), RRPFWIIRR-amide (RR-9) and PRFRWRIRI-amide (PI-9), are able to induce segregation of anionic lipids from zwitterionic lipids, as shown by changes in the phase transition properties of lipid mixtures detected by differential scanning calorimetry and freeze fracture electron microscopy. The relative Minimal Inhibitory Concentration (MIC) of these three peptides against several strains of Gram positive bacteria correlated well with the extent to which the lipid composition of the bacterial membrane facilitated peptide-induced clustering of anionic lipids. The lower activity of these three peptides against Gram negative bacteria could be explained by the retention of these peptides in the LPS layer. The membrane morphologies produced by PR-9 as well as by a cathelicidin fragment, KR-12 that had previously been shown to induce anionic lipid clustering, was directly visualized using freeze fracture electron microscopy. This work shows the insensitivity of phase segregation to the specific arrangement of the cationic charges in the peptide sequence as well as to their tendency to form different secondary structures. It also establishes the role of anionic lipid clustering in the presence of zwitterionic lipids in determining antimicrobial selectivity.
    Full-text · Article · Mar 2010 · Biochimica et Biophysica Acta
Show more