title: The effects of LPS on the activity of Trp-containing antimicrobial peptides against Gram-negative bacteria and endotoxin neutralization.
abstract: A series of synthesized Trp-containing antimicrobial peptides showed significantly different antimicrobial activity against Gram-negative bacteria despite having similar components and amino acid sequences and the same net positive charge and hydrophobicity. Lipopolysaccharide (LPS) in the outer membrane is a permeability barrier to prevent antimicrobial peptides from crossing into Gram-negative bacteria. We investigated the interaction of five Trp-containing peptides, I1W, I4W, L5W, L11W and L12W, with LPS using circular dichroism (CD), IR spectroscopy, isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements and confocal laser scanning microscopy, to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Our data indicate that I1W and I4W penetrated the LPS layer and killed Gram-negative bacteria by a "self-promoted uptake" pathway in which the peptides first approach LPS by electrostatic forces and then dissociate LPS micelle. This process results in disorganization of the LPS leaflet and promotes the ability of the peptide to cross the outer membrane into the inner membrane and disrupt the cytoplasmic membrane. Although L5W, L11W and L12W strongly bind to LPS bilayers and depolarize bacterial cytoplasmic membranes, similar to I1W and I4W, they are unable to destabilize LPS aggregates and traverse through the tightly packed LPS molecules. This study increases our understanding of the mechanism of action of these peptides in the LPS outer membrane and will help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes.
  
  
  CONCLUSIONS
  Tryptophan (Trp) residues show a strong preference for the interfacial region of biological membranes, and this property endows Trp-containing peptides with the unique ability to interact with the surface of bacterial cell membranes. In this manuscript, we report the membrane interaction of Trp-containing peptide to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Based on the data collected, we propose a molecular mechanism for the peptide-LPS interactions that allows the peptides to traverse or prevents them from transversing the LPS layer and the target inner membrane. The data should help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes.
authors.affiliation: Faculty of Life Science, Liaoning Normal University, Dalian 116081, China; Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Dalian 116081, China. Electronic address: djshang@lnnu.edu.cn.
authors.forename: Dejing
authors.initials: D
authors.lastname: Shang
authors.affiliation: Faculty of Life Science, Liaoning Normal University, Dalian 116081, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Faculty of Life Science, Liaoning Normal University, Dalian 116081, China; Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Dalian 116081, China.
authors.forename: Weibing
authors.initials: W
authors.lastname: Dong
authors.affiliation: Faculty of Life Science, Liaoning Normal University, Dalian 116081, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Liang
authors.affiliation: Faculty of Life Science, Liaoning Normal University, Dalian 116081, China.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Bi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002942:Circular Dichroism; D004731:Endotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D000067996:RAW 264.7 Cells; D013104:Spheroplasts / Q000187:drug effects; D013816:Thermodynamics; D014364:Tryptophan / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D004731:Endotoxins; D008070:Lipopolysaccharides; D010455:Peptides; D014364:Tryptophan
keywords: Antiendotoxin; Disassociation; Outer membrane; Permeability; Trp-containing peptides