Supramolecular Peptoid Structure Strengthens Complexation with Polyacrylic Acid Microgels.

We have studied the complexation between cationic antimicrobials and polyanionic microgels to create self-defensive surfaces that responsively resist bacterial colonization. An essential property is the stable sequestration of the loaded (complexed) antimicrobial within the microgel under a physiological ionic strength. Here, we assess the complexation strength between poly(acrylic acid) [PAA] microgels and a series of cationic peptoids that display supramolecular structures ranging from an oligomeric monomer to a tetramer. We follow changes in loaded microgel diameter with increasing [Na+] as a measure of the counterion doping level. Consistent with prior findings on colistin/PAA complexation, we find that a monomeric peptoid is fully released at ionic strengths well below physiological conditions, despite its +5 charge. In contrast, progressively higher degrees of peptoid supramolecular structure display progressively greater resistance to salting out, which we attribute to the greater entropic stability associated with the complexation of multimeric peptoid bundles.

Between good and evil: Complexation of the human cathelicidin LL-37 with nucleic acids.

The innate immune system provides a crucial first line of defense against invading pathogens attacking the body. As the only member of the human cathelicidin family, the antimicrobial peptide LL-37 has been shown to have antiviral, antifungal, and antibacterial properties. In complexation with nucleic acids, LL-37 is suggested to maintain its beneficial health effects while also acting as a condensation agent for the nucleic acid. Complexes formed by LL-37 and nucleic acids have been shown to be immunostimulatory with a positive impact on the human innate immune system. However, some studies also suggest that in some circumstances, LL-37/nucleic acid complexes may be a contributing factor to autoimmune disorders such as psoriasis and systemic lupus erythematosus. This review provides a comprehensive discussion of research highlighting the beneficial health effects of LL-37/nucleic acid complexes, as well as discussing observed detrimental effects. We will emphasize why it is important to investigate and elucidate structural characteristics, such as condensation patterns of nucleic acids within complexation, and their mechanisms of action, to shed light on the intricate physiological effects of LL-37 and the seemingly contradictory role of LL-37/nucleic acid complexes in the innate immune response.

Molecular Transport and Growth of Lipid Vesicles Exposed to Antimicrobial Peptides.

It is well-known that lipids constituting the cytoplasmic membrane undergo continuous reorganization to maintain the appropriate composition important for the integrity of the cell. The transport of lipids is controlled by mainly membrane proteins, but also spontaneous lipid transport between leaflets, lipid "flip-flop", occurs. These processes do not only occur spontaneously under equilibrium, but also promote structural rearrangements, morphological transitions, and growth processes. It has previously been shown that intravesicular lipid "flip-flop" and intervesicular lipid exchange under equilibrium can be deduced indirectly from contrast variation time-resolved small-angle neutron scattering (TR-SANS) where the molecules are "tagged" using hydrogen/deuterium (H/D) substitution. In this work, we show that this technique can be extended to simultaneously detect changes in the growth and the lipid "flip-flop" and exchange rates induced by a peptide additive on lipid vesicles consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), d-DMPC (1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), and small amounts of DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). Changes in the overall size were independently monitored using dynamic light scattering (DLS). We find that the antimicrobial peptide, indolicidin, accelerates lipid transport and additionally induces limited vesicular growth. Moreover, in TR-SANS experiments using partially labeled lipid mixtures to separately study the kinetics of the lipid components, we show that, whereas peptide addition affects both lipids similarly, DMPG exhibits faster kinetics. We find that vesicular growth is mainly associated with peptide-mediated lipid reorganization that only slightly affects the overall exchange kinetics. This is confirmed by a TR-SANS experiment of vesicles preincubated with peptide showing that after pre-equilibration the kinetics are only slightly slower.

Impact of antimicrobial peptides on E. coli-mimicking lipid model membranes: correlating structural and dynamic effects using scattering methods.

The mechanism of action of antimicrobial peptides (AMPs) has been debated over many years, and various models have been proposed. In this work we combine small angle X-ray/neutron scattering (SAXS/SANS) techniques to systematically study the effect of AMPs on the cytoplasmic membrane of Escherichia coli bacteria using a simplified model system of 4 : 1 DMPE : DMPG ([1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine] : [1,2-dimyristoyl-sn-glycero-3-phospho-(10-rac-glycerol)]) phospholipid unilamellar vesicles. The studied antimicrobial peptides aurein 1.2, indolicidin, LL-37, lacticin Q and colistin vary in size, charge, degree of helicity and origin. The peptides insert into the bilayer to various degrees, and are found to accelerate the dynamics of phospholipids significantly as seen by time resolved SANS (TR-SANS) measurements, with the exception of colistin that is suggested to rather interact with lipopolysaccharides (LPS) on the outer membrane of E. coli. We compare these results with earlier published data on model systems based on PC-lipids (phosphatidylcholines), showing comparable effect with regards to peptide insertion and effect on dynamics. However, model systems based on PE-lipids (phosphatidylethanolamine) are more prone to destabilisation upon addition of peptides, with formation of multilamellar structures and morphological changes. These properties of PE-vesicles lead to less conclusive results regarding peptide effect on structure and dynamics of the membrane.

Structure-Activity Study of an All-d Antimicrobial Octapeptide D2D.

The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH2 (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys5 and lys7, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 µg/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 1-8 showed that they likely fold into α-helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50-60%) inserts into the bilayer compared to D2D (~30-50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D.

Resolving the structural interactions between antimicrobial peptides and lipid membranes using small-angle scattering methods: the case of indolicidin.

Using small angle X-ray and neutron scattering (SAXS/SANS) and detailed theoretical modelling we have elucidated the structure of the antimicrobial peptide, indolicidin, and the interaction with model lipid membranes of different anionic lipid compositions mimicking typical charge densities found in the cytoplasmic membrane of bacteria. First, we show that indolicidin displays a predominantly disordered, random chain conformation in solution with a small fraction (≈1%) of fiber-like nanostructures that are not dissolved at higher temperatures. The peptide is shown to strongly interact with the membranes at all charge densities without significantly perturbing the lipid bilayer structure. Instead, the results show that indolicidin inserts into the outer leaflet of the lipid vesicles causing a reduced local order of the lipid packing. This result is supported by an observed change in the melting point of the lipids upon addition of the peptide, as seen by differential scanning calorimetry experiments. The peptide does not to our observation affect the thickness of the membrane or form distinct structural pores in the membrane at physiologically relevant concentrations as has been previously suggested as an important mode of action. Finally, using sophisticated contrast variation SANS, we show that the peptide does not affect the random lateral distribution of anionic lipids in the membrane. Together, these results demonstrate that the structural aspects of the mode of action of antimicrobial peptides can be elucidated in detail using SAS techniques with liposomes as model systems.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of morbidity and mortality in patients with cystic fibrosis, necessitating treatment with inhaled antibiotics. Pf phage is a filamentous bacteriophage produced by Pa that has been reported to act as a structural element in Pa biofilms. Pf presence has been associated with resistance to antibiotics and poor outcomes in cystic fibrosis, though the underlying mechanisms are unclear. Here, we have investigated how Pf phages and sputum biopolymers impede antibiotic diffusion using human sputum samples and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf phages and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf phages in sputum reduce the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf phages and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Accelerating Lipid Flip-Flop at Low Concentrations: A General Mechanism for Membrane Binding Peptides.

We report a physicochemical investigation of the lipid transport properties of model lipid membranes in the presence of the antimicrobial peptide indolicidin through comparisons of experimental SANS/SAXS scattering techniques to fully atomistic molecular dynamics simulations. In agreement with the experiment, we show that upon peripheral binding of the peptides, even at low concentrations, lipid flip-flop dynamics is greatly accelerated. Computer modeling elucidates the interplay between structural changes and lipid dynamics induced by peptides and proposes a mechanism for the mode of action of antimicrobial peptides, assessing the major role of entropy for the catalysis of the flipping events. The mechanism introduced here is universal for all peptides with preferential peripheral binding to the membrane as it does not depend on the specific amino acid sequence.

Real-Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label-Free Imaging with Optical Diffraction Tomography.

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug-resistant bacteria. As a mimic of AMPs, peptoids with N-substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure-activity relationship (SAR) of a library of indole side chain-containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label-free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real-time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance-breaking novel antibiotic drug.

Stability of Nanopeptides: Structure and Molecular Exchange of Self-assembled Peptide Fibers.

Often nanostructures formed by self-assembly of small molecules based on hydrophobic interactions are rather unstable, causing morphological changes or even dissolution when exposed to changes in aqueous media. In contrast, peptides offer precise control of the nanostructure through a range of molecular interactions where physical stability can be engineered in and, to a certain extent, decoupled from size via rational design. Here, we investigate a family of peptides that form beta-sheet nanofibers and demonstrate a remarkable physical stability even after attachment of poly(ethylene glycol). We employed small-angle neutron/X-ray scattering, circular dichroism spectroscopy, and molecular dynamics simulation techniques to investigate the detailed nanostructure, stability, and molecular exchange. The results for the most stable sequence did not reveal any structural alterations or unimer exchange for temperatures up to 85 °C in the biologically relevant pH range. Only under severe mechanical perturbation (i.e., tip sonication) would the fibers break up, which is reflected in a very high activation barrier for unimer exchange of ∼320 kJ/mol extracted from simulations. The results give important insight into the relation between molecular structure and stability of peptide nanostructure that is important for, e.g., biomedical applications.

Upregulating Human Cathelicidin Antimicrobial Peptide LL-37 Expression May Prevent Severe COVID-19 Inflammatory Responses and Reduce Microthrombosis.

COVID-19 is characterized by hyperactivation by inflammatory cytokines and recruitment of macrophages, neutrophils, and other immune cells, all hallmarks of a strong inflammatory response that can lead to severe complications and multi-organ damage. Mortality in COVID-19 patients is associated with a high prevalence of neutrophil extracellular trap (NET) formation and microthrombosis that are exacerbated by hyperglycemia, diabetes, and old age. SARS-CoV-2 infection in humans and non-human primates have revealed long-term neurological consequences of COVID-19, possibly concomitant with the formation of Lewy bodies in the brain and invasion of the nervous system via the olfactory bulb. In this paper, we review the relevance of the human cathelicidin LL-37 in SARS-CoV-2 infections. LL-37 is an immunomodulatory, host defense peptide with direct anti-SARS-CoV-2 activity, and pleiotropic effects on the inflammatory response, neovascularization, Lewy body formation, and pancreatic islet cell function. The bioactive form of vitamin D and a number of other compounds induce LL-37 expression and one might predict its upregulation, could reduce the prevalence of severe COVID-19. We hypothesize upregulation of LL-37 will act therapeutically, facilitating efficient NET clearance by macrophages, speeding endothelial repair after inflammatory tissue damage, preventing α-synuclein aggregation, and supporting blood-glucose level stabilization by facilitating insulin release and islet ß-cell neogenesis. In addition, it has been postulated that LL-37 can directly bind the S1 domain of SARS-CoV-2, mask angiotensin converting enzyme 2 (ACE2) receptors, and limit SARS-CoV-2 infection. Purposeful upregulation of LL-37 could also serve as a preventative and therapeutic strategy for SARS-CoV-2 infections.

Efficacy of Cathelicidin-Mimetic Antimicrobial Peptoids against Staphylococcus aureus.

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30 min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo. Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6 µM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8 days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections. IMPORTANCE Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death. These antimicrobial peptoids show efficient killing of bacteria both as a biofilm and as free bacteria, often doing so in less than 30 min. As such, these antimicrobials have the potential to alleviate the burden that Staphylococcus infections have on the health care system and cause better outcomes for infected patients.

Self-Assembly of Antimicrobial Peptoids Impacts Their Biological Effects on ESKAPE Bacterial Pathogens.

Antimicrobial peptides (AMPs) are promising pharmaceutical candidates for the prevention and treatment of infections caused by multidrug-resistant ESKAPE pathogens, which are responsible for the majority of hospital-acquired infections. Clinical translation of AMPs has been limited, in part by apparent toxicity on systemic dosing and by instability arising from susceptibility to proteolysis. Peptoids (sequence-specific oligo-N-substituted glycines) resist proteolytic digestion and thus are of value as AMP mimics. Only a few natural AMPs such as LL-37 and polymyxin self-assemble in solution; whether antimicrobial peptoids mimic these properties has been unknown. Here, we examine the antibacterial efficacy and dynamic self-assembly in aqueous media of eight peptoid mimics of cationic AMPs designed to self-assemble and two nonassembling controls. These amphipathic peptoids self-assembled in different ways, as determined by small-angle X-ray scattering; some adopt helical bundles, while others form core-shell ellipsoidal or worm-like micelles. Interestingly, many of these peptoid assemblies show promising antibacterial, antibiofilm activity in vitro in media, under host-mimicking conditions and antiabscess activity in vivo. While self-assembly correlated overall with antibacterial efficacy, this correlation was imperfect. Certain self-assembled morphologies seem better-suited for antibacterial activity. In particular, a peptoid exhibiting a high fraction of long, worm-like micelles showed reduced antibacterial, antibiofilm, and antiabscess activity against ESKAPE pathogens compared with peptoids that form ellipsoidal or bundled assemblies. This is the first report of self-assembling peptoid antibacterials with activity against in vivo biofilm-like infections relevant to clinical medicine.

Anti-persister and Anti-biofilm Activity of Self-Assembled Antimicrobial Peptoid Ellipsoidal Micelles.

Although persister cells are the root cause of resistance development and relapse of chronic infections, more attention has been focused on developing antimicrobial agents against resistant bacterial strains than on developing anti-persister agents. Frustratingly, the global preclinical antibacterial pipeline does not include any anti-persister drug. Therefore, the central point of this work is to explore antimicrobial peptidomimetics called peptoids (sequence-specific oligo-N-substituted glycines) as a new class of anti-persister drugs. In this study, we demonstrate that one particular antimicrobial peptoid, the sequence-specific pentamer TM5, is active against planktonic persister cells and sterilizes biofilms formed by both Gram-negative and Gram-positive bacteria. Moreover, we demonstrate the potential of TM5 to inhibit cytokine production induced by lipopolysaccharides from Gram-negative bacteria. We anticipate that this work can pave the way to the development of new anti-persister agents based on antimicrobial peptoids of this class to simultaneously help address the crisis of bacterial resistance and reduce the occurrence of the relapse of chronic infections.

Beyond structural models for the mode of action: How natural antimicrobial peptides affect lipid transport.

HYPOTHESIS: Most textbook models for antimicrobial peptides (AMP) mode of action are focused on structural effects and pore formation in lipid membranes, while these deformations have been shown to require high concentrations of peptide bound to the membrane. Even insertion of low amounts of peptides in the membrane is hypothesized to affect the transmembrane transport of lipids, which may play a key role in the peptide effect on membranes. EXPERIMENTS: Here we combine state-of-the-art small angle X-ray/neutron scattering (SAXS/SANS) techniques to systematically study the effect of a broad selection of natural AMPs on lipid membranes. Our approach enables us to relate the structural interactions, effects on lipid exchange processes, and thermodynamic parameters, directly in the same model system. FINDINGS: The studied peptides, indolicidin, aurein 1.2, magainin II, cecropin A and LL-37 all cause a general acceleration of essential lipid transport processes, without necessarily altering the overall structure of the lipid membranes or creating organized pore-like structures. We observe rapid scrambling of the lipid composition associated with enhanced lipid transport which may trigger lethal signaling processes and enhance ion transport. The reported membrane effects provide a plausible canonical mechanism of AMP-membrane interaction and can reconcile many of the previously observed effects of AMPs on bacterial membranes.

Lipid membrane interactions of self-assembling antimicrobial nanofibers: effect of PEGylation.

Supramolecular assembly and PEGylation (attachment of a polyethylene glycol polymer chain) of peptides can be an effective strategy to develop antimicrobial peptides with increased stability, antimicrobial efficacy and hemocompatibility. However, how the self-assembly properties and PEGylation affect their lipid membrane interaction is still an unanswered question. In this work, we use state-of-the-art small angle X-ray and neutron scattering (SAXS/SANS) together with neutron reflectometry (NR) to study the membrane interaction of a series of multidomain peptides, with and without PEGylation, known to self-assemble into nanofibers. Our approach allows us to study both how the structure of the peptide and the membrane are affected by the peptide-lipid interactions. When comparing self-assembled peptides with monomeric peptides that are not able to undergo assembly due to shorter chain length, we found that the nanofibers interact more strongly with the membrane. They were found to insert into the core of the membrane as well as to absorb as intact fibres on the surface. Based on the presented results, PEGylation of the multidomain peptides leads to a slight net decrease in the membrane interaction, while the distribution of the peptide at the interface is similar to the non-PEGylated peptides. Based on the structural information, we showed that nanofibers were partially disrupted upon interaction with phospholipid membranes. This is in contrast with the considerable physical stability of the peptide in solution, which is desirable for an extended in vivo circulation time.

Halogenation as a tool to tune antimicrobial activity of peptoids.

Antimicrobial peptides have attracted considerable interest as potential new class of antibiotics against multi-drug resistant bacteria. However, their therapeutic potential is limited, in part due to susceptibility towards enzymatic degradation and low bioavailability. Peptoids (oligomers of N-substituted glycines) demonstrate proteolytic stability and better bioavailability than corresponding peptides while in many cases retaining antibacterial activity. In this study, we synthesized a library of 36 peptoids containing fluorine, chlorine, bromine and iodine atoms, which vary by length and level of halogen substitution in position 4 of the phenyl rings. As we observed a clear correlation between halogenation of an inactive model peptoid and its increased antimicrobial activity, we designed chlorinated and brominated analogues of a known peptoid and its shorter counterpart. Short brominated analogues displayed up to 32-fold increase of the activity against S. aureus and 16- to 64-fold against E. coli and P. aeruginosa alongside reduced cytotoxicity. The biological effect of halogens seems to be linked to the relative hydrophobicity and self-assembly properties of the compounds. By small angle X-ray scattering (SAXS) we have demontrated how the self-assembled structures are dependent on the size of the halogen, degree of substitution and length of the peptoid, and correlated these features to their activity.

Helicity Modulation Improves the Selectivity of Antimicrobial Peptoids.

The modulation of conformational flexibility in antimicrobial peptides (AMPs) has been investigated as a strategy to improve their efficacy against bacterial pathogens while reducing their toxicity. Here, we synthesized a library of helicity-modulated antimicrobial peptoids by the position-specific incorporation of helix-inducing monomers. The peptoids displayed minimal variations in hydrophobicity, which permitted the specific assessment of the effect of conformational differences on antimicrobial activity and selectivity. Among the moderately helical peptoids, the most dramatic increase in selectivity was observed in peptoid 17, providing more than a 20-fold increase compared to fully helical peptoid 1. Peptoid 17 had potent broad-spectrum antimicrobial activity that included clinically isolated multi-drug-resistant pathogens. Compared to pexiganan AMP, 17 showed superior metabolic stability, which could potentially reduce the dosage needed, alleviating toxicity. Dye-uptake assays and high-resolution imaging revealed that the antimicrobial activity of 17 was, as with many AMPs, mainly due to membrane disruption. However, the high selectivity of 17 reflected its unique conformational characteristics, with differential interactions between bacterial and erythrocyte membranes. Our results suggest a way to distinguish different membrane compositions solely by helicity modulation, thereby improving the selectivity toward bacterial cells with the maintenance of potent and broad-spectrum activity.

A biophysical study of the interactions between the antimicrobial peptide indolicidin and lipid model systems.

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.