Abstract:Disruption of cell membranes is a fundamental host defence response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits pro… Show more
“…These results are in excellent agreement with the corresponding antimicrobial activity observed in the single-cell experiments. In light of our previous studies with SLBs 23 , and the fact that the bienA peptides are hemolytic 23 , both our single-cell and single-GUV studies indicate a membranolytic mechanism of action for these peptides, with bienA11 being the most potent of the set. Figure 5 A summary of the membranolytic activity of the bien peptides on populations of bacterial membrane-mimicking lipid vesicles.…”
Section: Resultssupporting
confidence: 62%
“…This is equivalent to the survival fraction measured via colony forming unit (CFU) bulk assays and we therefore used this as a quantitative metric for the inhibitory efficacy of the peptides 30 . The bienA11 and bienA10 (hemolytic 23 ) peptides were clearly the most potent in our assay, with a survival fraction of less than 5% (Fig. 2 A).…”
Section: Resultsmentioning
confidence: 88%
“…3 B, cells treated with bienA10 and bienA11 showed a 20% increase in length on average before cell death, whereas with the bienK series cells doubled in length (on average) before death. This is possibly linked to the membrane activity of the bienK peptides, which were found in supported lipid bilayer (SLB) experiments to disrupt the outer leaflet of lipid bilayers 23 ; these peptides may be exploiting weaknesses in the integrity of the membrane during the complex division process. However, there was significant variability in the response to each of the bienK peptides, and not all cells showed the same phenotype.…”
Section: Resultsmentioning
confidence: 99%
“…We recently reported the development of a novel set of ultrashort (≤ 11 amino acids) peptides, based on a minimal amphipathic helix with bi nary en coding (bien) by arginine and leucine, that showed antibacterial efficacy 23 . We refer to these compounds as “peptide” or “polypeptide” antibiotics, based on historical terminology used by Hancock 24 , also considering that these are synthetic peptides rather than natural AMPs 25 .…”
Section: Introductionmentioning
confidence: 99%
“…An alanine at the mutation position (“bienA”) facilitated a deep insertion of the helix in lipid membranes, and the formation of circular pores in (anionic) supported lipid bilayers (SLBs). However, a lysine at the same position (“bienK”) led to a shallower insertion of the helix and the formation of fractal membrane ruptures in the SLBs (peptide sequences are provided in Table 1 ) 23 . To probe the connection between the biological (cellular) and physico-chemical (membranolytic) properties of these new peptides, here we study the interaction of the bienA and bienK peptides with hundreds of individual Escherichia coli cells using the microfluidic “mother-machine” device 26 , and in parallel investigate their interactions with model membranes in a bespoke assay on hundreds of giant unilamellar vesicles (GUVs) 27 .…”
Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions—their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure–activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide’s physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.
“…These results are in excellent agreement with the corresponding antimicrobial activity observed in the single-cell experiments. In light of our previous studies with SLBs 23 , and the fact that the bienA peptides are hemolytic 23 , both our single-cell and single-GUV studies indicate a membranolytic mechanism of action for these peptides, with bienA11 being the most potent of the set. Figure 5 A summary of the membranolytic activity of the bien peptides on populations of bacterial membrane-mimicking lipid vesicles.…”
Section: Resultssupporting
confidence: 62%
“…This is equivalent to the survival fraction measured via colony forming unit (CFU) bulk assays and we therefore used this as a quantitative metric for the inhibitory efficacy of the peptides 30 . The bienA11 and bienA10 (hemolytic 23 ) peptides were clearly the most potent in our assay, with a survival fraction of less than 5% (Fig. 2 A).…”
Section: Resultsmentioning
confidence: 88%
“…3 B, cells treated with bienA10 and bienA11 showed a 20% increase in length on average before cell death, whereas with the bienK series cells doubled in length (on average) before death. This is possibly linked to the membrane activity of the bienK peptides, which were found in supported lipid bilayer (SLB) experiments to disrupt the outer leaflet of lipid bilayers 23 ; these peptides may be exploiting weaknesses in the integrity of the membrane during the complex division process. However, there was significant variability in the response to each of the bienK peptides, and not all cells showed the same phenotype.…”
Section: Resultsmentioning
confidence: 99%
“…We recently reported the development of a novel set of ultrashort (≤ 11 amino acids) peptides, based on a minimal amphipathic helix with bi nary en coding (bien) by arginine and leucine, that showed antibacterial efficacy 23 . We refer to these compounds as “peptide” or “polypeptide” antibiotics, based on historical terminology used by Hancock 24 , also considering that these are synthetic peptides rather than natural AMPs 25 .…”
Section: Introductionmentioning
confidence: 99%
“…An alanine at the mutation position (“bienA”) facilitated a deep insertion of the helix in lipid membranes, and the formation of circular pores in (anionic) supported lipid bilayers (SLBs). However, a lysine at the same position (“bienK”) led to a shallower insertion of the helix and the formation of fractal membrane ruptures in the SLBs (peptide sequences are provided in Table 1 ) 23 . To probe the connection between the biological (cellular) and physico-chemical (membranolytic) properties of these new peptides, here we study the interaction of the bienA and bienK peptides with hundreds of individual Escherichia coli cells using the microfluidic “mother-machine” device 26 , and in parallel investigate their interactions with model membranes in a bespoke assay on hundreds of giant unilamellar vesicles (GUVs) 27 .…”
Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions—their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure–activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide’s physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.
Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm À 1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions.
Chemistry has the power to endow supramolecular nanostructures with new biomedically relevant functions. Here it is reported that DNA nanostructures modified with cholesterol tags disrupt bacterial membranes to cause microbial cell death. The lipidated DNA nanostructures bind more readily to cholesterol‐free bacterial membranes than to cholesterol‐rich, eukaryotic membranes. These highly negatively charged, lipidated DNA nanostructures cause bacterial cell death by rupturing membranes. Strikingly, killing is mediated by clusters of barrel‐shaped nanostructures that adhere to the membrane without the involvement of expected bilayer‐puncturing barrels. These DNA nanomaterials may inspire the development of polymeric or small‐molecule antibacterial agents that mimic the principles of selective binding and rupturing to help combat antimicrobial resistance.
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