Design,expression, and characterization of the hybrid antimicrobial peptide T‐catesbeianin‐1 based on FyuA

The overuse of antibiotics has resulted in the emergence of antibiotic‐resistant bacteria, which presents an urgent need for new antimicrobial agents. At present, antimicrobial peptides have attracted a great deal of attention from researchers. However, antimicrobial peptides often affect a broad range of microorganisms, including the normal flora in a host organism. In the present study, we designed a novel hybrid antimicrobial peptide, expressed the hybrid peptide, and studied its specific target. The hybrid peptide, named T‐catesbeianin‐1, which includes the FyuA‐binding domain of pesticin and the peptide catesbeianin‐1, was designed and expressed in Pichia pastoris X‐33. Then, we determined the antimicrobial activity, cytotoxicity, and specific target of the peptide. T‐catesbeianin‐1 has strong antimicrobial activity and binds to FyuA to inhibit or kill Escherichia coli present in clinical specimens and mixed‐species culture. In summary, these findings suggested that T‐catesbeianin‐1 might be promising and specific antibiotic agent for therapeutic application against fyuA⁺ E. coli.     

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

Background

Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.

Results

The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.

Conclusions

The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.     

A novel RNA‐binding peptide regulates the establishment of the Medicago truncatula–Sinorhizobium meliloti nitrogen‐fixing symbiosis

Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen‐fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so‐called nodulin genes are induced, including large families that encode small peptides. Using a three‐hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA‐binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single‐stranded RNA‐binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA‐binding peptides.     

Insight into the bovine milk peptide LPcin‐YK3 selection in the proteolytic system of Lactobacillus species

Antimicrobial peptides are class of small, positively charged peptides known for their broad‐spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin‐YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100‐fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide‐binding protein A and LPcin‐YK3 peptide. Given that degradation related to the LPcin‐YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin‐YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide‐binding protein A in respect of LPcin‐YK3 peptide.     

Bioengineering and functional characterization of arenicin shortened analogs with enhanced antibacterial activity and cell selectivity

New bioengineering approaches are required for development of more active and less toxic antimicrobial peptides. In this study we used β‐hairpin antimicrobial peptide arenicin‐1 as a template for design of more potent antimicrobials. In particular, six shortened 17‐residue analogs were obtained by recombinant expression in Escherichia coli. Besides, we have introduced the second disulfide bridge by analogy with the structure of tachyplesins. As a result, a number of analogs with enhanced activity and cell selectivity were developed. In comparison with arenicin‐1, which acts on cell membranes with low selectivity, the most potent and promising its analog termed ALP1 possessed two‐fold higher antibacterial activity and did not affect viability of mammalian cells at concentration up to 50 μM. The therapeutic index of ALP1 against both Gram‐positive and Gram‐negative bacteria was significantly increased compared with that of arenicin‐1 while the mechanism of action remained the same. Like arenicin‐1, the analog rapidly disrupt membranes of both stationary and exponential phase bacterial cells and effectively kills multidrug‐resistant Gram‐negative bacteria. Furthermore, ALP1 was shown to bind DNA in vitro at a ratio of 1:1 (w/w). The circular dichroism spectra demonstrated that secondary structures of the shortened analogs were similar to that of arenicin‐1 in water solution, but significantly differed in membrane‐mimicking environments. This work shows that a strand length is one of the key parameters affecting cell selectivity of β‐hairpin antimicrobial peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Investigating the nucleic acid interactions and antimicrobial mechanism of buforin II

Buforin II (BF2) is an antimicrobial peptide that is hypothesized to kill bacteria by entering cells and binding nucleic acids. To further investigate this proposed mechanism, we used computer modeling and experimental measurements to consider the interactions between BF2 and DNA. Computational and experimental results imply that the peptide forms specific interactions with DNA. Moreover, we observe a general correlation between DNA affinity and antimicrobial activity for a series of BF2 variants. Thus, our results support the proposed mechanism for BF2 and provide a useful approach for evaluating the nucleic acid interactions of other antimicrobial peptides.     

The unstructured domain of colicin N kills Escherichia coli

Bacteria often produce toxins which kill competing bacteria. Colicins, produced by and toxic to Escherichia coli bacteria are three‐domain proteins so efficient that one molecule can kill a cell. The C‐terminal domain carries the lethal activity and the central domain is required for surface receptor binding. The N‐terminal domain, required for translocation across the outer membrane, is always intrinsically unstructured. It has always been assumed therefore that the C‐terminal cytotoxic domain is required for the bactericidal activity. Here we report the unexpected finding that in isolation, the 90‐residue unstructured N‐terminal domain of colicin N is cytotoxic. Furthermore it causes ion leakage from cells but, unlike known antimicrobial peptides (AMPs) with this property, shows no membrane binding behaviour. Finally, its activity remains strictly dependent upon the same receptor proteins (OmpF and TolA) used by full‐length colicin N. This mechanism of rapid membrane disruption, via receptor mediated binding of a soluble peptide, may reveal a new target for the development of highly specific antibacterials.     

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2‐amino acid with either two 2‐naphthyl‐methylene or two para‐CF₃‐benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5‐fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9‐fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC‐5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide‐based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Ribosomal binding and antibacterial activity of ethylene glycol‐bridged apidaecin Api137 and oncocin Onc112 conjugates

Recent surveillance data on antimicrobial resistance predict the beginning of the post‐antibiotic era with pan‐resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline‐rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram‐negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin‐type) or the assembly of the 50S subunit (apidaecin‐type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112‐conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)‐carboxyfluorescein‐labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112‐conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP‐susceptible and ‐resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin‐ and oncocin‐type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of antimicrobial peptides of neutrophils on tumor and normal cells in culture

We performed a comparative study of effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine bactenecin 5 (Bac5) on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic beta-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic properties. Bac5 belongs to the group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a non-lytic mechanism. We have shown that while PG1 exerts distinct and fast cytotoxic effects on most of used tumor cells being slightly less toxic for nontransformed host cell, the proline-rich Bac5 is much less cytotoxic for all the cells tested. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as had been previously established for human defensins by Panyutich et al., 1995). Pre-incubation of PG1 with alpha1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of alpha1-antitrypsin was reduced after interaction of the serpin with PG1 (not with Bac5). Confocal microscopy experiments allowed to monitor the internalization of fluorescent labeled (by BODIPY FL) peptides into target cells and their intracellular distribution. Bac5-BODIPY (at 5 microM) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations was also able to enter the cells without significant damage to them. The comparative study of the kinetics of the peptides uptake into the target cells and the influence of low temperature, energy-depletion and endocytosis inhibitors on the process of the internalization of the peptides into the cells was carried out using flow cytometry.     

Recent Advances in Stereoselective Drug Targeting

Reviewed here are some recent examples of medically important protein targets for which stereoselective drugs have been identified. These include heat shock protein 90 (Hsp90) inhibitors as anticancer agents; transient receptor potential vanilloid type 1 antagonists as new analgesics; stereospecific inhibition of human mutT homolog MTH1 for cancer treatment; the stereoselective binding of R‐ and S‐propranolol by the α1–acid glycoprotein transporter; metallohelical complexes that are nonpeptide α‐helical mimetics that enantioselectively target Aβ amyloid for the treatment of Alzheimer's disease; metallohelical assemblies with promising antimicrobial activity that enantioselectively target DNA of resistant bacteria; nonpeptide α‐helical metallohelices that target the DNA of cisplatin‐resistant cancer cells; diastereomeric selectivity of phenanthriplatin‐guanine adducts; and phenazine biosynthetic enzyme active sites that can host both enantiomers of a racemic ligand simultaneously. Chirality 27:589–597, 2015. © 2015 Wiley Periodicals, Inc.     

Specificity of anti-peptide antibodies elicited against synthetic peptides mimicking conserved regions of HIV1 envelope glycoprotein

Comparison of HIV1_Bru and HIV2_Rod external envelope glycoprotein sequences enabled us to select ten highly conserved peptide sequences. The corresponding peptides were chemically synthesized, then coupled to bovine serum albumin before injection in rabbits. Although all peptides were immunogenic, only antibodies directed against peptides P1 (amino acid residues 33–55), P22 (418–462), P8 (487–508) and P21 (487–534) were able to interact with significant affinity (K_0.5 about 10⁻⁶ and 10⁻⁸ M) with the native glycoprotein by radioimmunoassay. Noteworthy was the capacity of anti-P1 antibodies to also recognize the glycoprotein of HIV2. Anti-peptide antibodies were tested for their ability to interfere with the gp120-CD4 interaction, membrane fusion and virus replication. Preincubation of gp 120 with antibodies directed to the region previously described as the putative CD4-binding site, P22 (418–462), did not abolish gp120 binding to CD4-positive cells.     

The continuing story of class IIa bacteriocins.

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.     

鱼精蛋白抑菌机理及在食品防腐中的应用

鱼精蛋白是一类天然的阳离子抗菌肽,具有广谱抑菌活性。鱼精蛋白主要是通过破坏细菌的细胞壁、细胞膜及改变细胞的渗透性等途径抑制甚至杀死细菌细胞。在鱼精蛋白抑制细菌的同时,细菌也产生多种机制对抗鱼精蛋白。温度、pH、阳离子和EDTA等多种理化因子影响鱼精蛋白对细菌的抑制效果。由于鱼精蛋白在抑菌防腐方面的众多优势,目前已成为非常有发展前景的食品防腐剂。     

Purification,characterization and application of a novel antimicrobial peptide from Andrias davidianus blood

The Andrias davidianus has been known as a traditional Chinese medicine for a long time. Its blood is considered as a waste or by‐product of the meat production industry. Although there are reports on isolation of the antimicrobial peptides from different resources, there are no reports of their isolation from A. davidianus blood. In this work, an antimicrobial peptide, andricin B, was isolated from the blood of A. davidianus by an innovative method in which the magnetic liposome adsorption was combined with reversed‐phase high‐performance liquid chromatography. The structure, antimicrobial activity and safety of andricin B were further investigated. Amino acid sequence was determined by N‐terminal sequencing and found to be Gly‐Leu‐Thr‐Arg‐Leu‐Phe‐Ser‐Val‐Ile‐Lys. Circular dichroism (CD) spectra and prediction of three‐dimensional structure by bioinformatics software suggested the presence of a well‐defined random coil conformation. Andricin B was found to be active against all bacteria tested in this study as well as some fungi. The minimum inhibitory concentrations (MICs) were in the range 8–64 μg ml^?1. Moreover, the haemolytic testing also suggested that andricin B could be considered safe at the MICs. Finally, andricin B was shown to inhibit the growth of Staphylococcus aureus in the cooked meat of A. davidianus. This study shows that andricin B is a promising novel antimicrobial peptide that may provide further insights towards the development of new drugs.

Significance and Impact of the Study

This is the pioneer study on screening and isolation of antimicrobial peptide from the blood of Andrias davidianus. Here, we have developed a novel method by combining magnetic liposomes adsorption with reversed‐phase high‐performance liquid chromatography to purify and screen the antimicrobial peptides. From this screen, we identified a novel antimicrobial peptide which we name as andricin B. Andricin B is unique as it checks the growth of both Gram‐positive and Gram‐negative bacteria as well as few fungal species.     

Inhibitory effects and mechanisms of physiological conditions on the activity of enantiomeric forms of an α-helical antibacterial peptide against bacteria

Enantiomeric amphipathic α-helical antibacterial peptides were synthesized and their biophysical and biological properties under different physiological conditions were studied. In the absence of physiological factors, the l- and d-peptides exhibited similar antimicrobial activities against a broad spectrum of bacteria, even against clinical isolates with resistance to traditional antibiotics. However, in the presence of NaCl, CaCl₂ or human serum albumin (HSA) at physiological concentrations, the enantiomers revealed bacterium-species dependent attenuations in antibacterial activity. In the presence of salts the electrostatic interaction between the peptides and the biomembrane was inhibited. Salts, especially CaCl_2, weakened the ability of the peptides to permeabilize the outer membrane of Gram-negative bacteria, as determined by a 1-N-phenylnaphthylamine uptake assay. HSA exhibited variable inhibitory effects on the activity of the peptides when incubated with different bacterial strains. The peptides showed different binding association abilities to HSA at different molar ratios, regardless of their chirality, resulting in reduced peptide biological activity. The d-peptide performed better than its l-enantiomer in all conditions tested because of its resistance to proteolysis, and may therefore represent a promising candidate for development as a therapeutic agent.     

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two alpha/beta-antimicrobial peptides

We have previously shown that two synthetic antimicrobial peptides with alternating alpha- and beta-amino acid residues, designated simply as alpha/beta-peptide I and alpha/beta-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of alpha/beta-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by alpha/beta-peptide II. The alpha/beta-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of alpha/beta-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. alpha/beta-Peptide II is more effective in this regard because, unlike alpha/beta-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although alpha/beta-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why alpha/beta-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of alpha/beta-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.