Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers

Biochemistry. 2000 Nov 28;39(47):14504-14. doi: 10.1021/bi0011173.

Abstract

Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles. Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in animal models. Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL. All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents. None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis. As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells. Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes. Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Infective Agents / administration & dosage
  • Anti-Infective Agents / chemical synthesis
  • Anti-Infective Agents / chemistry*
  • Anti-Infective Agents / pharmacology
  • Antimicrobial Cationic Peptides / administration & dosage
  • Antimicrobial Cationic Peptides / chemical synthesis
  • Antimicrobial Cationic Peptides / chemistry*
  • Antimicrobial Cationic Peptides / pharmacology
  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / metabolism
  • Cell Membrane Permeability / drug effects
  • Circular Dichroism
  • DNA-Binding Proteins / chemical synthesis
  • DNA-Binding Proteins / chemistry
  • Disease Models, Animal
  • Endotoxins / antagonists & inhibitors
  • Female
  • Hemolysin Proteins / pharmacology
  • Horseshoe Crabs
  • Humans
  • Injections, Intraperitoneal
  • Lipopolysaccharides / chemistry*
  • Lipopolysaccharides / metabolism
  • Liposomes / chemistry
  • Liposomes / metabolism
  • Mice
  • Microbial Sensitivity Tests
  • Neutropenia / microbiology
  • Neutropenia / mortality
  • Neutropenia / prevention & control
  • Peptides, Cyclic / chemical synthesis
  • Peptides, Cyclic / chemistry
  • Phospholipids / chemistry*
  • Protein Binding / drug effects
  • Protein Engineering
  • Protein Isoforms / chemical synthesis
  • Protein Isoforms / chemistry
  • Protein Isoforms / pharmacology
  • Protein Structure, Secondary
  • Sequence Homology, Amino Acid*
  • Structure-Activity Relationship

Substances

  • Anti-Infective Agents
  • Antimicrobial Cationic Peptides
  • Bacterial Outer Membrane Proteins
  • DNA-Binding Proteins
  • Endotoxins
  • Hemolysin Proteins
  • Lipopolysaccharides
  • Liposomes
  • Peptides, Cyclic
  • Phospholipids
  • Protein Isoforms
  • tachyplesin peptide, Tachypleus tridentatus
  • polyphemusin I