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Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides
Authors:Epand Raquel F  Mowery Brendan P  Lee Sarah E  Stahl Shannon S  Lehrer Robert I  Gellman Samuel H  Epand Richard M
Institution:1 Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8N 3Z5
2 Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA
3 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
Abstract: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.
Keywords:LUV  large unilamellar vesicle  DOPE  1  2-dioleoyl-sn-glycero-3-phosphoethanolamine  DOPG  1  2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]  CL  cardiolipin  DOPC  1  2-dioleoyl-sn-glycero-3-phosphocholine  ITC  isothermal titration calorimetry  ANTS  8-aminonaphthalene-1  3  6  trisulfonic acid  DPX  p-xylene-bis-pyridinium bromide  SUV  small unilamellar vesicle  DSC  differential scanning calorimetry  DPPE  1  2-dipalmitoyl-sn-glycero-3-phosphoethanolamine  LPS  lipopolysaccharide  LTA  lipoteichoic acid  OM  outer membrane  IM  inner membrane  ONPG  d-galactoside" target="_blank">o-nitrophenyl-3-d-galactoside  MIC  minimal inhibitory concentration  MBC  minimal bactericidal concentration  MLV  multilamellar vesicle  POPE  1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine  TSB  tryptic soy broth
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