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

Lee E, Kim JK, Jeon D, Jeong KW, Shin A, Kim Y - Sci Rep (2015)

Bottom Line: PapN exhibited significant broad-spectrum antibacterial activities without cytotoxicity.The PapN series peptides permeabilized bacterial membranes less effectively than papiliocin, showing no antibacterial activities in an hour.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea.

ABSTRACT
A cecropin-like peptide, papiliocin, isolated from the swallowtail butterfly Papilio xuthus, possesses high selectivity against gram-negative bacteria. Since Trp(2) and Phe(5) are highly conserved residues in cecropin-like peptides, we investigated the role of Trp(2) and Phe(5) in antibacterial activity. Substitution of Trp(2) and Phe(5) in papiliocin with Ala (papiliocin-2A and papiliocin-5A) revealed that Trp(2) 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 Arg(1)-Ala(22) 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 Trp(2) and Phe(5) 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.

No MeSH data available.


Related in: MedlinePlus

Dose-dependent toxic activity of papiliocin and its analogs toward (A) human RBCs and (B) mouse macrophage-derived RAW264.7 cells. All values represent the means ± standard deviations of three independent experiments.
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f4: Dose-dependent toxic activity of papiliocin and its analogs toward (A) human RBCs and (B) mouse macrophage-derived RAW264.7 cells. All values represent the means ± standard deviations of three independent experiments.

Mentions: We examined the cytotoxicity against human erythrocytes. Figure 4A shows the dose-dependent curves of hemolytic activities of the peptides. Papiliocin and the PapN series do not show hemolytic activity, even at 100 μM, while PapC produced 49% hemolytic activity, even at a low concentration of 6.25 μM. Next, we examined the cytotoxicity of the peptides against RAW264.7 macrophage cells. We assessed the effects of the peptides on cell growth by measuring mitochondrial reduction of MTT to a colored product in live cells. Melittin exhibited high cytotoxicity at its MIC, whereas papiliocin and the PapN series were not toxic at their MICs (Fig. 4A,B). The survival rates of RAW264.7 cells in the presence of 50 μM papiliocin and PapN were approximately 76.1% and 86.1%, respectively. All the PapN peptides were less toxic than papiliocin. On the other hand, PapC produced high cytotoxicity against RAW264.7 cells, even at low concentrations. The survival rate of RAW264.7 cells was only 44.2% in the presence of 10 μM PapC. Notably, the C-terminal helix region is a critical determinant of the cytotoxicity of papiliocin. Substituting Ala for Trp2 or Phe5 in papiliocin resulted in more than a 20% decrease in cytotoxicity against RAW264.7 cells at 100 uM papiliocin. In case of PapN-2A, Pap-5A, and Pap-2A5A, the cell survival rates at a peptide concentration of 100 μM were approximately 90% or more. Thus, Trp2 and Phe5 are important for the cytotoxic activity of papiliocin toward RAW264.7 cells.


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

Lee E, Kim JK, Jeon D, Jeong KW, Shin A, Kim Y - Sci Rep (2015)

Dose-dependent toxic activity of papiliocin and its analogs toward (A) human RBCs and (B) mouse macrophage-derived RAW264.7 cells. All values represent the means ± standard deviations of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496781&req=5

f4: Dose-dependent toxic activity of papiliocin and its analogs toward (A) human RBCs and (B) mouse macrophage-derived RAW264.7 cells. All values represent the means ± standard deviations of three independent experiments.
Mentions: We examined the cytotoxicity against human erythrocytes. Figure 4A shows the dose-dependent curves of hemolytic activities of the peptides. Papiliocin and the PapN series do not show hemolytic activity, even at 100 μM, while PapC produced 49% hemolytic activity, even at a low concentration of 6.25 μM. Next, we examined the cytotoxicity of the peptides against RAW264.7 macrophage cells. We assessed the effects of the peptides on cell growth by measuring mitochondrial reduction of MTT to a colored product in live cells. Melittin exhibited high cytotoxicity at its MIC, whereas papiliocin and the PapN series were not toxic at their MICs (Fig. 4A,B). The survival rates of RAW264.7 cells in the presence of 50 μM papiliocin and PapN were approximately 76.1% and 86.1%, respectively. All the PapN peptides were less toxic than papiliocin. On the other hand, PapC produced high cytotoxicity against RAW264.7 cells, even at low concentrations. The survival rate of RAW264.7 cells was only 44.2% in the presence of 10 μM PapC. Notably, the C-terminal helix region is a critical determinant of the cytotoxicity of papiliocin. Substituting Ala for Trp2 or Phe5 in papiliocin resulted in more than a 20% decrease in cytotoxicity against RAW264.7 cells at 100 uM papiliocin. In case of PapN-2A, Pap-5A, and Pap-2A5A, the cell survival rates at a peptide concentration of 100 μM were approximately 90% or more. Thus, Trp2 and Phe5 are important for the cytotoxic activity of papiliocin toward RAW264.7 cells.

Bottom Line: PapN exhibited significant broad-spectrum antibacterial activities without cytotoxicity.The PapN series peptides permeabilized bacterial membranes less effectively than papiliocin, showing no antibacterial activities in an hour.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea.

ABSTRACT
A cecropin-like peptide, papiliocin, isolated from the swallowtail butterfly Papilio xuthus, possesses high selectivity against gram-negative bacteria. Since Trp(2) and Phe(5) are highly conserved residues in cecropin-like peptides, we investigated the role of Trp(2) and Phe(5) in antibacterial activity. Substitution of Trp(2) and Phe(5) in papiliocin with Ala (papiliocin-2A and papiliocin-5A) revealed that Trp(2) 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 Arg(1)-Ala(22) 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 Trp(2) and Phe(5) 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.

No MeSH data available.


Related in: MedlinePlus