Interaction Of Trp-containing Antimicrobial Peptides With LPS And Antimicrobial Activity

The septic shock has been a great threat to human health which is caused by Gram-negative bacteria. To analyze the interactions of antimicrobial peptides with lipopolysaccharide(LPS), the effects of LPS on the antimicrobial activities of cationic antimicrobial peptides were investigated in this paper.L-K6 is a modified peptide of temporin-1CEb, a native antimicrobial peptide from the skin secretions of Rana chensinensis. In our previous studies, seven novel Trp-containing antimicrobial peptides were synthesized based on L-K6 by replacing Ile or Leu with Trp at different positions on the hydrophobic face of L-K6. These antimicrobial peptides with 6 net positive charges, showed good antibacterial activity against E. coli and other three kinds of Gram-negative bacteria. In this paper, the effects of LPS on the antibacterial activity of these antimicrobial peptides were investigated. The results showed that LPS could inhibit the antibacterial activity of these antimicrobial peptides, especially L11 W and L12 W. These Trp-containing antimicrobial peptides showed strong effect of membrane depolarization on the protoplasts of E. coli, which was similar to that of Gram-positive bacteria Staphylococcus aureus lacking the LPS outer membrane, but low effect on intact E. coli. LPS showed different effect on the membrane depolarization of these seven antimicrobial peptides, especially on L12 W. Similar to the effect of depolarization, calcein-release experiments further confirmed LPS could reduce the membrane damage caused by these peptides. Laser confocal microscopy experiments was used to confirm the antimicrobial peptides were capable of killing the bacteria by disrupting the cell membranes of E. coli, because the Trp residues have high hydrophobicity, which makes them easily insert into the bacterial membrane.In this paper the antimicrobial peptides interaction with LPS were also studied by CD, isothermal titration calorimetry(ITC), dynamic light scattering(DLS) and FITC-LPS depolymerization experiments. CD spectra showed that the Trp-containing antimicrobial peptides presented a random coil structure in SP buffer solution, but in the presence of LPS, they exhibited an α- helical structure. ITC experiments showed the process of the binding of peptides to LPS was an exothermic process, which means the positive charge peptide bind with the negatively charged LPS by electrostatic interaction. Zeta-potential experiments also showed that these seven antimicrobial peptides can interact with LPS, which have charge compensation. DLS and FITC-LPS depolymerization experiments showed that after binding of antimicrobial peptides with LPS, LPS aggregates could be depolymerized, the polymerization state of LPS molecules became smaller particle size, due to the penetration of antimicrobial peptides. I1WL5 W could completely eliminate the LPS aggregates macromolecules.In summary, although LPS inhibits the antimicrobial activity of some Trp-containing antimicrobial peptides by reducing their membrane permeability, several antimicrobial peptides can play a bactericidal effect by their interaction with LPS, and result in penetrating the outer membrane to reach the inner membrane.