Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of peptides on eukaryotic cells.Effects of peptides on HaCaT cells and erythrocytes in absence and presence of 20% human serum. The MTT-assay (upper panel) was used to measure viability of HaCaT keratinocytes in the presence of KNK10 peptides with variable W tagging. In the assay, MTT is modified into a dye, blue formazan, by enzymes associated to metabolic activity. The absorbance of the dye was measured at 550 nm. Cell permeabilizing effects of the indicated peptides (middle panel) were measured by the LDH-based TOX-7 kit. Hemolytic effects of the indicated peptides are also shown (lower panel). Cells were incubated with peptides at 60 µM, while 2% Triton X-100 served as positive control. The absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis (mean values are presented, n = 3). (For MTT and LDH, the difference between tagged and non-tagged peptides is statistically significant in the absence of serum (P<0.001, one way ANOVA), whereas the difference in the presence of serum is not statistically significant. For hemolysis, the difference between tagged and non-tagged peptides is not statistically significant.)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2667214&req=5

pone-0005285-g002: Effect of peptides on eukaryotic cells.Effects of peptides on HaCaT cells and erythrocytes in absence and presence of 20% human serum. The MTT-assay (upper panel) was used to measure viability of HaCaT keratinocytes in the presence of KNK10 peptides with variable W tagging. In the assay, MTT is modified into a dye, blue formazan, by enzymes associated to metabolic activity. The absorbance of the dye was measured at 550 nm. Cell permeabilizing effects of the indicated peptides (middle panel) were measured by the LDH-based TOX-7 kit. Hemolytic effects of the indicated peptides are also shown (lower panel). Cells were incubated with peptides at 60 µM, while 2% Triton X-100 served as positive control. The absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis (mean values are presented, n = 3). (For MTT and LDH, the difference between tagged and non-tagged peptides is statistically significant in the absence of serum (P<0.001, one way ANOVA), whereas the difference in the presence of serum is not statistically significant. For hemolysis, the difference between tagged and non-tagged peptides is not statistically significant.)

Mentions: The non-tagged peptides show no hemolysis above that of the negative control (Figure 1 and Figure 2. Tagging with WWW results in little, if any, increase in hemolysis, whereas that of the longer WWWWW results in a slightly increased hemolysis. This effect is concentration dependent, with hemolysis for KNK10-WWWWW being comparable to, or lower than, that of LL-37 (Figure 1B). Similarly, tagging with WWWWW, but not WWW, results in an increased cell permeabilization monitored by MTT and LDH assays. In the presence of serum, on the other hand, also the WWWWW-tagged peptide displays no detectable permeabilization with either LDH release or MTT assay (Figure 2). Again, similar results were obtained for the F-tagged peptides (Figure S1B).


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)

Effect of peptides on eukaryotic cells.Effects of peptides on HaCaT cells and erythrocytes in absence and presence of 20% human serum. The MTT-assay (upper panel) was used to measure viability of HaCaT keratinocytes in the presence of KNK10 peptides with variable W tagging. In the assay, MTT is modified into a dye, blue formazan, by enzymes associated to metabolic activity. The absorbance of the dye was measured at 550 nm. Cell permeabilizing effects of the indicated peptides (middle panel) were measured by the LDH-based TOX-7 kit. Hemolytic effects of the indicated peptides are also shown (lower panel). Cells were incubated with peptides at 60 µM, while 2% Triton X-100 served as positive control. The absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis (mean values are presented, n = 3). (For MTT and LDH, the difference between tagged and non-tagged peptides is statistically significant in the absence of serum (P<0.001, one way ANOVA), whereas the difference in the presence of serum is not statistically significant. For hemolysis, the difference between tagged and non-tagged peptides is not statistically significant.)
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005285-g002: Effect of peptides on eukaryotic cells.Effects of peptides on HaCaT cells and erythrocytes in absence and presence of 20% human serum. The MTT-assay (upper panel) was used to measure viability of HaCaT keratinocytes in the presence of KNK10 peptides with variable W tagging. In the assay, MTT is modified into a dye, blue formazan, by enzymes associated to metabolic activity. The absorbance of the dye was measured at 550 nm. Cell permeabilizing effects of the indicated peptides (middle panel) were measured by the LDH-based TOX-7 kit. Hemolytic effects of the indicated peptides are also shown (lower panel). Cells were incubated with peptides at 60 µM, while 2% Triton X-100 served as positive control. The absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis (mean values are presented, n = 3). (For MTT and LDH, the difference between tagged and non-tagged peptides is statistically significant in the absence of serum (P<0.001, one way ANOVA), whereas the difference in the presence of serum is not statistically significant. For hemolysis, the difference between tagged and non-tagged peptides is not statistically significant.)
Mentions: The non-tagged peptides show no hemolysis above that of the negative control (Figure 1 and Figure 2. Tagging with WWW results in little, if any, increase in hemolysis, whereas that of the longer WWWWW results in a slightly increased hemolysis. This effect is concentration dependent, with hemolysis for KNK10-WWWWW being comparable to, or lower than, that of LL-37 (Figure 1B). Similarly, tagging with WWWWW, but not WWW, results in an increased cell permeabilization monitored by MTT and LDH assays. In the presence of serum, on the other hand, also the WWWWW-tagged peptide displays no detectable permeabilization with either LDH release or MTT assay (Figure 2). Again, similar results were obtained for the F-tagged peptides (Figure S1B).

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