Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

Biometals. 2014 Oct;27(5):949-56. doi: 10.1007/s10534-014-9760-5. Epub 2014 Jun 25.

Abstract

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antimicrobial Cationic Peptides / chemistry
  • Antimicrobial Cationic Peptides / genetics
  • Antimicrobial Cationic Peptides / pharmacology
  • Burkholderia / drug effects*
  • Burkholderia / metabolism
  • Burkholderia / ultrastructure
  • Burkholderia pseudomallei / drug effects*
  • Burkholderia pseudomallei / metabolism
  • Burkholderia pseudomallei / ultrastructure
  • Cattle
  • Cell Membrane / drug effects
  • Freeze Fracturing
  • Hemolysis / drug effects
  • Humans
  • Lactoferrin / chemistry*
  • Lactoferrin / genetics
  • Lactoferrin / pharmacology*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / pharmacology
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / pharmacology
  • Species Specificity

Substances

  • Antimicrobial Cationic Peptides
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • lactoferrampin
  • lactoferricin (17-30)
  • Lactoferrin