Functional Roles of Aromatic Residues and Helices of Papiliocin in its Antimicrobial and Anti-inflammatory Activities

Abstract

A cecropin-like peptide, papiliocin, isolated from the swallowtail butterfly Papilio xuthus, possesses high selectivity against gram-negative bacteria. Since Trp2 and Phe5 are highly conserved residues in cecropin-like peptides, we investigated the role of Trp2 and Phe5 in antibacterial activity. Substitution of Trp2 and Phe5 in papiliocin with Ala (papiliocin-2A and papiliocin-5A) revealed that Trp2 is a key residue in its antibacterial activities. In order to understand the structural requirements for papiliocin function and to design shorter, but more potent, peptide antibiotics, we designed papiliocin constructs, PapN (residues Arg1-Ala22 from the N-terminal amphipathic helix). PapN exhibited significant broad-spectrum antibacterial activities without cytotoxicity. Bactericidal kinetics of peptides against E.coli showed that papiliocin completely and rapidly killed E.coli in less than 10 minutes at 2× MIC concentration, while papiliocin-2A and papiliocin-5A killed four times more slowly than papiliocin. The PapN series peptides permeabilized bacterial membranes less effectively than papiliocin, showing no antibacterial activities in an hour. The results imply that the Trp2 and Phe5 in the amphipathic N-terminal helix are important in the rapid permeabilization of the gram-negative bacterial membrane. The hydrophobic C-terminal residues permeabilize the hydrophobic bacterial cell membrane synergistically with these aromatic residues, providing selectivity against gram-negative bacteria.

Structure-Activity Relationships of Peptide Antibiotics with Improved Bacterial Cell Selectivity of Pseudin

“…We performed phase-sensitive 2D experiments, including total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) using time-proportional phase incrementation. 8,9 50 and 80 ms MLEV-17 spin-lock mixing pulses were used for TOCSY experiments and for NOESY experiments, mixing times of 150 ms and 250 ms were used. The 3 J HN coupling constants were measured from the DQF-COSY spectra.…”

Structure-Activity Relationship of the N-terminal Helix Analog of Papiliocin, PapN

“…Bactericidal kinetics of peptides against E.coli showed that papiliocin completely and rapidly killed E.coli in less than 10 minutes at 2 × MIC concentration, while PapN permeabilized bacterial membranes less effectively than papiliocin. 10 The results imply that the Trp 2 and Phe 5 in the amphipathic N-terminal helix are important in the rapid permeabilization of the gram-negative bacterial membrane. In this study, to gain further insight into the structure-activity relationships, we determined the 3D-structures of PapN in 300 mM DPC micelles and investigated the interactions between DPC micelles and PapN.…”

“…9 In order to understand the structural requirements for papiliocin function and to design shorter and potent peptide antibiotics, we designed papiliocin analog, PapN (residues Arg 1 -Ala 22 from the N-terminal amphipathic helix). 10 PapN exhibited significant broad-spectrum antibacterial activities without cytotoxicity. Bactericidal kinetics of peptides against E.coli showed that papiliocin completely and rapidly killed E.coli in less than 10 minutes at 2 × MIC concentration, while PapN permeabilized bacterial membranes less effectively than papiliocin.…”

Short Antimicrobial Peptides Exhibiting Antibacterial and Anti‐Inflammatory Activities Derived from the N‐Terminal Helix of Papiliocin

“…Previous studies showed that papiliocin containing an N‐terminal helix sequence exhibited high levels of antibacterial activity comparable to that of melittin . Additionally, the aromatic residues Trp2 and Phe5 located in the N‐terminal α‐helix are crucial for papiliocin antibacterial activity.…”

“…Additionally, the aromatic residues Trp2 and Phe5 located in the N‐terminal α‐helix are crucial for papiliocin antibacterial activity. The N‐terminal helix region alone harbors activity, while only C‐terminal region did not show antibacterial activity . Furthermore, the C‐terminal region of intact papiliocin is crucial for its high selectivity against Gram‐negative bacteria .…”