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End-tagging of ultra-short antimicrobial peptides by W/F stretches to facilitate bacterial killing.

Pasupuleti M, Schmidtchen A, Chalupka A, Ringstad L, Malmsten M - PLoS ONE (2009)

Bottom Line: Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus.Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum.Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase.

View Article: PubMed Central - PubMed

Affiliation: Section of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden.

ABSTRACT

Background: Due to increasing resistance development among bacteria, antimicrobial peptides (AMPs), are receiving increased attention. Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Additionally, short and proteolytically stable AMPs are desired to maximize bioavailability and therapeutic versatility.

Methodology and principal findings: A facile approach is demonstrated for reaching high potency of ultra-short antimicrobal peptides through end-tagging with W and F stretches. Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Through end-tagging, potency and salt resistance could be maintained down to 4-7 amino acids in the hydrophilic template peptide. Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum. Quantitatively, the most potent peptides investigated displayed bactericidal effects comparable to, or in excess of, that of the benchmark antimicrobial peptide LL-37. The higher bactericidal potency of the tagged peptides correlated to a higher degree of binding to bacteria, and resulting bacterial wall rupture. Analogously, tagging enhanced peptide-induced rupture of liposomes, particularly anionic ones. Additionally, end-tagging facilitated binding to bacterial lipopolysaccharide, both effects probably contributing to the selectivity displayed by these peptides between bacteria and eukaryotic cells. Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo for pig skin infected by S. aureus and E. coli.

Conclusions/significance: End-tagging by hydrophobic amino acid stretches may be employed to enhance bactericidal potency also of ultra-short AMPs at maintained limited toxicity. The approach is of general applicability, and facilitates straightforward synthesis of hydrophobically modified AMPs without the need for post-peptide synthesis modifications.

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Peptide interaction with bacteria and LPS.(A) Permeabilizing effects of peptides on bacteria. E. coli was incubated with KNK7, KNK10, and the indicated W-modified variants (all at 30 µM) in buffer at physiological salt (0.15 M NaCl) for 2 h at 37°C, after which permeabilization was assessed using the impermeant probe FITC. The upper images in each row are Nomarski Differential Interference Contrast images, while the lower show FITC fluorescence of bacteria. (B) LPS and heparin binding abilities of the KNK7, KNK10 and the indicated W-modified variants peptides.
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pone-0005285-g003: Peptide interaction with bacteria and LPS.(A) Permeabilizing effects of peptides on bacteria. E. coli was incubated with KNK7, KNK10, and the indicated W-modified variants (all at 30 µM) in buffer at physiological salt (0.15 M NaCl) for 2 h at 37°C, after which permeabilization was assessed using the impermeant probe FITC. The upper images in each row are Nomarski Differential Interference Contrast images, while the lower show FITC fluorescence of bacteria. (B) LPS and heparin binding abilities of the KNK7, KNK10 and the indicated W-modified variants peptides.

Mentions: As can be seen in Figure 3A, the increased bactericidal potency of the W-tagged peptides correlates to a higher permeabilization of bacteria. In analogy, results from anionic liposomes composed of either a bacteria-mimicking lipid mixture (DOPE/DOPG) or lipid extract from E. coli, showed tagged peptides to be much more potent in causing membrane rupture and liposome leakage that the corresponding non-tagged ones (Figure 4A). For both these lipid mixtures, rapid and extensive leakage induction was observed with the tagged peptides (Figure 4B). As with bacterial killing, peptide-induced liposome leakage increased with increasing peptide concentration and hydrophobic tag length, and was partially reduced at high ionic strength, the salt inactivation decreasing with increasing hydrophobic tag length. In analogy to the bactericidal and cytotoxicity results, liposome leakage induction by the tagged peptides is substantial for both negatively charged (“bacterial”) lipid mixtures investigated, but substantially lower for zwitterionic (“eukaryotic”) DOPC liposomes, particularly at high ionic strength. Additionally, W-tagging was found to facilitate binding of both KNK10 and KNK7 to LPS (and heparin) also at high ionic strength (Figure 3B), an effect which can be completely reversed through addition of heparin, acting as an anionic competitor to LPS for the tagged peptides (results not shown).


End-tagging of ultra-short antimicrobial peptides by W/F stretches to facilitate bacterial killing.

Pasupuleti M, Schmidtchen A, Chalupka A, Ringstad L, Malmsten M - PLoS ONE (2009)

Peptide interaction with bacteria and LPS.(A) Permeabilizing effects of peptides on bacteria. E. coli was incubated with KNK7, KNK10, and the indicated W-modified variants (all at 30 µM) in buffer at physiological salt (0.15 M NaCl) for 2 h at 37°C, after which permeabilization was assessed using the impermeant probe FITC. The upper images in each row are Nomarski Differential Interference Contrast images, while the lower show FITC fluorescence of bacteria. (B) LPS and heparin binding abilities of the KNK7, KNK10 and the indicated W-modified variants peptides.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667214&req=5

pone-0005285-g003: Peptide interaction with bacteria and LPS.(A) Permeabilizing effects of peptides on bacteria. E. coli was incubated with KNK7, KNK10, and the indicated W-modified variants (all at 30 µM) in buffer at physiological salt (0.15 M NaCl) for 2 h at 37°C, after which permeabilization was assessed using the impermeant probe FITC. The upper images in each row are Nomarski Differential Interference Contrast images, while the lower show FITC fluorescence of bacteria. (B) LPS and heparin binding abilities of the KNK7, KNK10 and the indicated W-modified variants peptides.
Mentions: As can be seen in Figure 3A, the increased bactericidal potency of the W-tagged peptides correlates to a higher permeabilization of bacteria. In analogy, results from anionic liposomes composed of either a bacteria-mimicking lipid mixture (DOPE/DOPG) or lipid extract from E. coli, showed tagged peptides to be much more potent in causing membrane rupture and liposome leakage that the corresponding non-tagged ones (Figure 4A). For both these lipid mixtures, rapid and extensive leakage induction was observed with the tagged peptides (Figure 4B). As with bacterial killing, peptide-induced liposome leakage increased with increasing peptide concentration and hydrophobic tag length, and was partially reduced at high ionic strength, the salt inactivation decreasing with increasing hydrophobic tag length. In analogy to the bactericidal and cytotoxicity results, liposome leakage induction by the tagged peptides is substantial for both negatively charged (“bacterial”) lipid mixtures investigated, but substantially lower for zwitterionic (“eukaryotic”) DOPC liposomes, particularly at high ionic strength. Additionally, W-tagging was found to facilitate binding of both KNK10 and KNK7 to LPS (and heparin) also at high ionic strength (Figure 3B), an effect which can be completely reversed through addition of heparin, acting as an anionic competitor to LPS for the tagged peptides (results not shown).

Bottom Line: Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus.Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum.Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase.

View Article: PubMed Central - PubMed

Affiliation: Section of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden.

ABSTRACT

Background: Due to increasing resistance development among bacteria, antimicrobial peptides (AMPs), are receiving increased attention. Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Additionally, short and proteolytically stable AMPs are desired to maximize bioavailability and therapeutic versatility.

Methodology and principal findings: A facile approach is demonstrated for reaching high potency of ultra-short antimicrobal peptides through end-tagging with W and F stretches. Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Through end-tagging, potency and salt resistance could be maintained down to 4-7 amino acids in the hydrophilic template peptide. Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum. Quantitatively, the most potent peptides investigated displayed bactericidal effects comparable to, or in excess of, that of the benchmark antimicrobial peptide LL-37. The higher bactericidal potency of the tagged peptides correlated to a higher degree of binding to bacteria, and resulting bacterial wall rupture. Analogously, tagging enhanced peptide-induced rupture of liposomes, particularly anionic ones. Additionally, end-tagging facilitated binding to bacterial lipopolysaccharide, both effects probably contributing to the selectivity displayed by these peptides between bacteria and eukaryotic cells. Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo for pig skin infected by S. aureus and E. coli.

Conclusions/significance: End-tagging by hydrophobic amino acid stretches may be employed to enhance bactericidal potency also of ultra-short AMPs at maintained limited toxicity. The approach is of general applicability, and facilitates straightforward synthesis of hydrophobically modified AMPs without the need for post-peptide synthesis modifications.

Show MeSH
Related in: MedlinePlus