Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the "carpet-model" and thus, provide important insights into their antimicrobial mechanisms.     

Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity

RTA3 is an α-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of Escherichiacoli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E.coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides.     

Modulating short tryptophan- and arginine-rich peptides activity by substitution with histidine

BackgroundHigh antimicrobial efficacy of short tryptophan-and arginine-rich peptides makes them good candidates in the fight against pathogens. Substitution of tryptophan and arginine by histidine could be used to modulate the peptides efficacy by optimizing their structures.MethodsThe peptide (RRWWRWWRR), reported to showed good antimicrobial efficacy, was used as template, seven new analogs being designed substituting tryptophan or arginine with histidine. The peptides' efficacy was tested against E. coli, B. subtilis and S. aureus. The cytotoxicity and hemolytic effect were evaluated and the therapeutic index was inferred for each peptide. Atomic force microscopy and molecular simulation were used to analyze the effects of peptides on bacterial membrane.ResultsThe substitution of tryptophan by histidine proved to strongly modulate the antimicrobial activity, mainly by changing the peptide-to-membrane binding energy. The substitution of arginine has low effect on the antimicrobial efficacy. The presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides in some cases maintaining the same efficacy against bacteria. The peptides' antimicrobial activity was correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter.ConclusionThe results show that some of these peptides have the potential to become good candidates to fight against bacteria. The substitution by histidine proved to fine tune the therapeutic index allowing the optimization of the peptide structure mainly by changing its binding energy and 3D-hydrophobic moment.General significanceThe short tryptophan reach peptides therapeutic index can be maximized using the histidine substitution to optimize their structure.     

The lysine-peptoid hybrid LP5 maintain activity under physiological conditions and affects virulence gene expression in Staphylococcus aureus

The antimicrobial peptide, LP5, is a lysine-peptoid hybrid, with antimicrobial activity against clinically relevant bacteria. Here, we investigated how various environmental conditions affect the antimicrobial activity of LP5 against Staphylococcus aureus (S. aureus). We found that LP5 maintained activity under host physiological conditions of NaCl, MgCl₂ and pH. However, when exposed to serum, LP5 lost activity. Furthermore, when increasing NaCl concentration and lowering pH, the peptide showed reduces activity. When investigating the tolerance mechanisms of S. aureus toward antimicrobial peptides, we found that LP5 was protease resistant. However, the dltA and vraF genes, involved in reducing the net anionic charge of the bacterial cell envelope and sensing of antimicrobial peptides, respectively, played a role in the tolerance of S. aureus against LP5. In addition, the exposure of S. aureus to sub-inhibitory concentrations of LP5 affected the expression of the major virulence factors of S. aureus, revealing a potential as anti-virulence compound. Thus, these results show how environmental factors affect the peptide efficiency and further add to the knowledge on how the peptide affects S. aureus, which is crucial information for designing new peptides for optimizing antimicrobial therapy.     

Novel Method for Selection of Antimicrobial Peptides from a Phage Display Library by Use of Bacterial Magnetic Particles

Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from “Magnetospirillum magneticum” strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 × 10¹⁰ PFU of the library phage (complexity, 2.7 × 10⁹) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species.     

Molecular mechanisms that govern the specificity of Sushi peptides for Gram-negative bacterial membrane lipids

Factor C-derived Sushi peptides (S1 and S3) have been shown to bind lipopolysaccharide (LPS) and inhibit the growth of Gram-negative bacteria but do not affect mammalian cells. On the premise that the composition of membrane phospholipids differs between the microbial and human cells, we studied the modes of interaction between S1 and S3 and the bacterial membrane phospholipids, POPG, in comparison to that with the mammalian cell membrane phospholipids, POPC and POPE. S1 exhibits specificity against POPG, suggesting its preference for bacterial anionic phospholipids, regardless of whether the phospholipids form vesicles in a solution or a monolayer on a solid surface. The specificity of the Sushi peptides for POPG is a consequence of the electrostatic and hydrophobic forces. The unsaturated nature of POPG confers fluidity to the lipid layer, and being in the proximity of LPS in the microenvironmental milieu, POPG probably enhances the insertion of the peptide-LPS complex into the bacterial inner membrane. Furthermore, during its interaction with POPG, the S1 peptide underwent a transition from random to alpha-helical coil, while S3 became a mixture of beta-sheet and alpha-helical structures. This differential structural change in the peptides could be responsible for their different modes of disruption of POPG vesicles. Conceivably, the selectivity for POPG spares the mammalian membranes from undesirable effects of antimicrobial peptides, which could be helpful in designing and developing a new generation of antibiotics and in offering some clues about the specific function of Factor C, a LPS biosensor.     

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.     

RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity

Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide β114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.     

Physical behavior of KR-12 peptide on solid surfaces and Langmuir-Blodgett lipid films: Complementary approaches to its antimicrobial mode against S. aureus

Biophysical characterization of antimicrobial peptides helps to understand the mechanistic aspects of their action. The physical behavior of the KR-12 antimicrobial peptide (e.g. orientation and changes in secondary structure), was analyzed after interactions with a Staphylococcus aureus membrane model and solid surfaces. We performed antimicrobial tests using Gram-positive S. aureus (ATCC 25923) bacteria. Moreover, Langmuir-Blodgett experiments showed that the synthetic peptide can disturb the lipidic membrane at a concentration lower than the Minimum Inhibitory Concentration, thus confirming that KR-12/lipid interactions are involved. Partially- and fully-deactivated KR-12 hybrid samples were obtained by physisorption and covalent immobilization in chitosan/silica and glyoxal-rich solid supports. The correlation of Langmuir-Blodgett data with the α-helix formation, followed by FTIR-ATR in a frozen-like state, and the antimicrobial activity showed the importance of these interactions and conformation changes on the first step action mode of this peptide. This is the first time that material science (immobilization in solid surfaces assisted by FTIR-ATR analysis in frozen-like state) and physical (Langmuir-Blodgett/Schaefer) approaches are combined for exploring mechanistic aspects of the primary action mode of the KR-12 antimicrobial peptide against S. aureus.     

Intestinal dysbiosis and reduced immunoglobulin-coated bacteria associated with coeliac disease in children

Background

NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn).

Results

In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against Staphylococcus aureus), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin) were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P.

Conclusions

NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane.     

Interactions of an anionic antimicrobial peptide with Staphylococcus aureus membranes

The antimicrobial activity of the anionic peptide, AP1 (GEQGALAQFGEWL), was investigated. AP1 was found to kill Staphylococcus aureus with an MLC of 3 mM and to induce maximal surface pressure changes of 3.8 mN m⁻¹ over 1200 s in monolayers formed from lipid extract of S. aureus membranes. FTIR spectroscopy showed the peptide to be α-helical (100%) in the presence of vesicles formed from this lipid extract and to induce increases in their fluidity (Δν circa 0.5 cm⁻¹). These combined data show that AP1 is able to function as an α-helical antimicrobial peptide against Gram-positive bacteria and suggest that the killing mechanism used by the peptide involves interactions with the membrane lipid headgroup region. Moreover, this killing mechanism differs strongly from that previously reported for AP1 against Gram-negative bacteria, indicating the importance of considering the effects of membrane lipid composition when investigating the structure/function relationships of antimicrobial peptides.     

Antifungal activity of C3a and C3a-derived peptides against Candida

Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys⁵⁷-Arg⁷⁷). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.     

Partition of antimicrobial D-L-α-cyclic peptides into bacterial model membranes

Fluorescence spectroscopy is used to characterize the partition of three second-generation D,L-α-cyclic peptides to two lipid model membranes. The peptides have proven antimicrobial activity, particularly against Gram positive bacteria, and the model membranes are formed of either with 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG) or its mixture with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), at a molar ratio of (1:1). The peptide's intrinsic fluorescence was used in the Steady State and/or Time Resolved Fluorescence Spectroscopy experiments, showing that the peptides bind to the membranes, and the extent of their partition is thereof quantified. The peptide-induced membrane leakage was followed using an encapsulated fluorescent dye.Overall, the partition is mainly driven by electrostatics, but also involves hydrophobic interactions. The introduction of a hydrocarbon tail in one of the residues of the parent peptide, CPR, adjacent to the tryptophan (Trp) residue, significantly improves the partition of the modified peptides, CPRT10 and CPRT14, to both membrane systems. Further, we show that the length of the tail is the main distinguishing factor for the extension of the partition process.The parent peptide induces very limited leakage, at odds with the peptides with tail, that promote fast leakage, increasing in most cases with peptide concentration, and being almost complete for the highest peptide concentration and negatively charged membranes.Overall, the results help the unravelling of the antimicrobial action of these peptides and are well in line with their proven high antimicrobial activity.     

Dynamics of the Antimicrobial Peptide PGLa Action on Escherichia coli Monitored by Atomic Force Microscopy

Summary Atomic force microscopy (AFM) images of living cells in physiological solution were used to monitor the different stages involved in the interaction between Escherichia coli and the antimicrobial peptide PGLa. Damage on bacterial membranes was observed in the past using standard electron microscopy; stiffness measurements and images scanned in physiological solution demonstrate the advantage of AFM for such studies. From force versus separation curve measurements it is possible to determine the variation of the cellular stiffness. PGLa action on components of the cell structure like the outer membrane, the bacterial pili, the peptidoglycan wall and the inner membrane was determined by the comparison of AFM images of bacteria before and after PGLa addition. The interaction of Escherichia coli with PGLa in the culture medium has two stages. The first is characterized by the loss of surface stiffness and the formation of micelles probably originating from the disruption of the outer membrane and the loss of the bacteria’s ability to adhere to the substrates. In the second stage there is further damage, which resulted in total cell rupture. AFM images of bacteria in air and surface roughness measurements were also used to estimate peptide damage.     

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa).The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition.We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.     

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes

A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk l-α-phosphatidylcholine (EYPC)/egg yolk l-α-phosphatidyl-dl-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-d-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.     

Membrane-active and DNA binding related double-action antimycobacterial mechanism of antimicrobial peptide W3R6 and its synthetic analogs

The emergence of multidrug- or extremely drug-resistant M. tuberculosis strains has made very few drugs available for current tuberculosis treatment. Antimicrobial peptides can be employed as a promising alternative strategy for TB treatment. Here, we designed and synthesized a series of peptide sequences based on the structure-activity relationships of natural sequences of antimicrobial peptides. The peptide W3R6 and its analogs were screened and found to have potent antimycobacterial activity against M. smegmatis, and no hemolytic activity against human erythrocytes. The evidence from the mechanism of action study indicated that W3R6 and its analogs can interact with the mycobacterial membrane in a lytic manner and form pores on the outer membrane of M. smegmatis. Significant colocalization of D-W3R6 with mycobacterial DNA was observed by confocal laser scanning microscopy and DNA retardation assays, which suggested that the antimycobacterial mechanism of action of the peptide was associated with the unprotected genomic DNA of M. smegmatis. In general, W3R6 and its analogs act on not only the mycobacterial membrane but also the genomic DNA in the cytoplasm, which makes it difficult for mycobacteria to generate resistance due to the peptides having two targets. In addition, the peptides can effectively eliminate M. smegmatis cells from infected macrophages. Our findings indicated that the antimicrobial peptide W3R6 could be a novel lead compound to overcome the threat from drug-resistant M. tuberculosis strains in the development of potent AMPs for TB therapeutic applications.     

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.