Interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)6(Arg), A6R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A6R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A6R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A6R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A6R exhibits antimicrobial activity without membrane lysis

Comparing Surfactant Structures at "Soft" and "Hard" Hydrophobic Materials: Not All Interfaces Are Equivalent.

The interfacial structures of a range of amphiphilic molecules are studied with both "soft" and "hard" hydrophobic substrates. Neutron reflection and quartz crystal microbalance with dissipation measurements highlight the differences between the adsorbed structures adopted by sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (C16TAB), and the "AM1" surface active peptide. At the soft siloxane/water interface, small molecular surfactants form loosely packed layers, with the hydrophobic tails penetrating into the oily layer, and an area per surfactant molecule that is significantly less than previously reported for the air/water interface. Neutron reflection measurements, supported by quartz crystal microbalance studies, indicate that for C16TAB, approximately 30 ± 8% of the alkyl tail penetrates into the poly(dimethylsiloxane) (PDMS) layer, whereas 20 ± 5% of the alkyl tail of SDS is located in the PDMS. For the engineered peptide surfactant AM1 (21 residues), it was found that one face of the α helix penetrated into the PDMS film. In contrast, penetration of the surfactant tails was not observed against hard solidlike hydrophobic surfaces made from octadecyltrichlorosilane (OTS) for any of the molecular species studied. At the OTS/water interface, C16TAB and SDS were seen to adsorb as larger aggregates and not as monolayers. Amphiphilic adsorption (amount, structural conformation) at the PDMS/water interface is shown to be different from that at both the air/water interface and the hard OTS/water interface, illustrating that interfacial structures cannot be predicted by the surfactant packing parameter alone. The bound PDMS layer is shown to be a useful proxy for the oil/water interface in surface and stabilization studies, with hydrophobic components of the molecules able to penetrate into the oily PDMS

Interactions of Non-steroidal Anti-inflammatory Drugs and Their Bismuth Analogues (BiNSAIDs) with Biological Membrane Mimics at Physiological pH.

Previous studies have demonstrated the potential for non-steroidal anti-inflammatory drugs (NSAIDs), in particular aspirin, to be used as chemopreventives for colorectal cancer; however, a range of unwanted gastrointestinal side effects limit their effectiveness. Due to the role of bismuth in the treatment of gastrointestinal disorders, it is hypothesized that bismuth-coordinated NSAIDs (BiNSAIDs) could be used to combat the gastrointestinal side effects of NSAIDs while still maintaining their chemopreventive potential. To further understand the biological activity of these compounds, the present study examined four NSAIDs, namely, tolfenamic acid (tolfH), aspirin (aspH), indomethacin (indoH), and mefenamic acid (mefH) and their analogous homoleptic BiNSAIDs ([Bi(L)3]n), to determine how these compounds interact with biological membrane mimics composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and cholesterol. Electrical impedance spectroscopy studies revealed that each of the NSAIDs and BiNSAIDs influenced membrane conductance, suggesting that temporary pore formation may play a key role in the previously observed cytotoxicity of tolfH and Bi(tolf)3. Quartz crystal microbalance with dissipation monitoring showed that all the compounds were able to interact with membrane mimics composed of solely POPC or POPC/cholesterol. Finally, neutron reflectometry studies showed changes in membrane thickness and composition. The location of the compounds within the bilayer could not be determined with certainty; however, a complex interplay of interactions governs the location of small molecules, such as NSAIDs, within lipid membranes. The charged nature of the parent NSAIDs means that interactions with the polar headgroup region are most likely with larger hydrophobic sections, potentially leading to deeper penetration

Comparing Surfactant Structures at "soft" and "hard" Hydrophobic Materials: Not All Interfaces Are Equivalent

The interfacial structures of a range of amphiphilic molecules are studied with both "soft" and "hard" hydrophobic substrates. Neutron reflection and quartz crystal microbalance with dissipation measurements highlight the differences between the adsorbed structures adopted by sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (C16TAB), and the "AM1" surface active peptide. At the soft siloxane/water interface, small molecular surfactants form loosely packed layers, with the hydrophobic tails penetrating into the oily layer, and an area per surfactant molecule that is significantly less than previously reported for the air/water interface. Neutron reflection measurements, supported by quartz crystal microbalance studies, indicate that for C16TAB, approximately 30 ± 8% of the alkyl tail penetrates into the poly(dimethylsiloxane) (PDMS) layer, whereas 20 ± 5% of the alkyl tail of SDS is located in the PDMS. For the engineered peptide surfactant AM1 (21 residues), it was found that one face of the α helix penetrated into the PDMS film. In contrast, penetration of the surfactant tails was not observed against hard solidlike hydrophobic surfaces made from octadecyltrichlorosilane (OTS) for any of the molecular species studied. At the OTS/water interface, C16TAB and SDS were seen to adsorb as larger aggregates and not as monolayers. Amphiphilic adsorption (amount, structural conformation) at the PDMS/water interface is shown to be different from that at both the air/water interface and the hard OTS/water interface, illustrating that interfacial structures cannot be predicted by the surfactant packing parameter alone. The bound PDMS layer is shown to be a useful proxy for the oil/water interface in surface and stabilization studies, with hydrophobic components of the molecules able to penetrate into the oily PDMS

QCM-D fingerprinting of membrane-active peptides

The increasing prevalence of antibiotic-resistant bacteria is becoming a public health crisis. Antimicrobial peptides (AMPs) are a promising solution, because bacterial resistance is less likely. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a versatile and valuable technique for investigation of these peptides. This article looks at the different approaches to the interpretation of QCM-D data, showing how to extract the maximum information from the data. Five AMPs of diverse charge, length and activity are used as case studies: caerin 1.1 wild-type, two caerin 1.1 mutants (Gly15Gly19-caerin 1.1 and Ala15Ala19-caerin 1.1), aurein 1.2 and oncocin. The interaction between the AMP and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane is analysed inter alia using frequency-dissipation plots (∆f-∆D plots) to ascertain the mechanism of action of the AMP. The ∆f-∆D plot can then be used to provide a fingerprint for the AMP-membrane interaction. Building up a database of these fingerprints for all known AMPs will enable the relationship between AMP structure and membrane activity to be better understood, hopefully leading to the future development of antibiotics without bacterial resistance.George A. McCubbin, Slavica Praporski, Stefania Piantavigna, Daniel Knappe, Ralf Hoffmann, John H. Bowie, Frances Separovic, Lisandra L. Marti

Insights into the interfacial structure-function of poly(ethylene glycol)-decorated peptide-stabilised nanoscale emulsions

The interfacial properties of nanoscale materials have profound influence on biodistribution and stability as well as the effectiveness of sophisticated surface-encoded properties such as active targeting to cell surface receptors. Tailorable nanocarrier emulsions (TNEs) are a novel class of oil-in-water emulsions stabilised by molecularly-engineered biosurfactants that permit single-pot stepwise surface modification with related polypeptides that may be chemically conjugated or genetically fused to biofunctional moieties. We have probed the structure and function of poly(ethylene glycol) (PEG) used to decorate TNEs in this way. The molecular weight of PEG decorating TNEs has considerable impact on the ζ-potential of the emulsion particles, related to differential interfacial thickness of the PEG layer as determined by X-ray reflectometry. By co-modifying TNEs with an antibody fragment, we show that the molecular weight and density of PEG governs the competing parameters of accessibility of the targeting moiety and of shielding the interface from non-specific interactions with the environment. The fundamental understanding of the molecular details of the PEG layer that we present provides valuable insights into the structure–function relationship for soft nanomaterial interfaces. This work therefore paves the way for further rational design of TNEs and other nanocarriers that must interact with their environment in controlled and predictable ways.Hossam H. Tayeb, Stefania Piantavigna, Christopher B. Howard, Amanda Nouwens, Stephen M. Mahler, Anton P. J. Middelberg, Lizhong He, Stephen A. Holt and Frank Sainsbur

Insights into the interfacial structure-function of poly(ethylene glycol)-decorated peptide-stabilised nanoscale emulsions.

The interfacial properties of nanoscale materials have profound influence on biodistribution and stability as well as the effectiveness of sophisticated surface-encoded properties such as active targeting to cell surface receptors. Tailorable nanocarrier emulsions (TNEs) are a novel class of oil-in-water emulsions stabilised by molecularly-engineered biosurfactants that permit single-pot stepwise surface modification with related polypeptides that may be chemically conjugated or genetically fused to biofunctional moieties. We have probed the structure and function of poly(ethylene glycol) (PEG) used to decorate TNEs in this way. The molecular weight of PEG decorating TNEs has considerable impact on the ζ-potential of the emulsion particles, related to differential interfacial thickness of the PEG layer as determined by X-ray reflectometry. By co-modifying TNEs with an antibody fragment, we show that the molecular weight and density of PEG governs the competing parameters of accessibility of the targeting moiety and of shielding the interface from non-specific interactions with the environment. The fundamental understanding of the molecular details of the PEG layer that we present provides valuable insights into the structure-function relationship for soft nanomaterial interfaces. This work therefore paves the way for further rational design of TNEs and other nanocarriers that must interact with their environment in controlled and predictable ways

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug Resistant Gram Negative Pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a KD of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin

Amyloid aggregation and membrane activity of the antimicrobial peptide uperin 3.5

Amyloid fibrils are highly ordered, b-sheet rich forms of aggregated peptides and proteins that are associated with a variety of pathological human disorders, including Alzheimer’s and Parkinson’s diseases. Amyloid fibril-forming peptides may be functionally related to antimicrobial peptides, despite differing significantly in sequence and structure. Specifically, their interaction with lipid membranes has mechanistic similarities. The 17-amino acid peptide uperin 3.5 (U3.5) from an Australian amphibian is antimicrobial and amyloidogenic. Using a quartz crystal microbalance, we investigated the interaction of U3.5 with artificial membranes and found that (i) the membrane interaction of U3.5 is independent of the peptide’s aggregation state, (ii) the presence of cholesterol in the membrane dramatically alters peptide–membrane interaction leading to a transmembrane pore-like arrangement of U3.5, and (iii) electrostatic interaction is important for the membrane activity of U3.5 whereby removal of the positive charge at position 7 of U3.5 enhanced its fibrillar aggregation and ablated its membrane interaction, i.e. there is an inverse relationship between the antimicrobial and amyloidogenic properties of U3.5.Lisandra L. Martin, Clemens Kubeil, Stefania Piantavigna, Tarun Tikkoo, Nicholas P. Gray, Torsten John, Antonio N. Calabrese, Yanqin Liu, Yuning Hong, Mohammed A. Hossain, Nitin Patil, Bernd Abel, Ralf Hoffmann, John H. Bowie, John A. Carve