Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.     

Identification and structural analysis of the antimicrobial domain in hipposin, a 51-mer antimicrobial peptide isolated from Atlantic halibut

Hipposin is a potent 51-mer antimicrobial peptide (AMP) from Atlantic halibut with sequence similarity to parasin (19-mer catfish AMP), buforin I (39-mer toad AMP), and buforin II (an active 21-mer fragment of buforin I), suggesting that the antimicrobial activity of these peptides might all be due to a common antimicrobial sequence motif. In order to identify the putative sequence motif, the antimicrobial activity of hipposin fragments against 20 different bacteria was compared to the activity of hipposin, parasin and buforin II. Neither parasin nor the 19-mer parasin-like fragment HIP(1-19) (differs from parasin in only three residues) that is derived from the N-terminal part (residues 1-19) of hipposin had marked antimicrobial activity. In contrast, the fragment HIP(16-36) (identical to buforin II) that is derived from the middle part of hipposin (residues 16-36) had such activity, indicating that this part of hipposin contained an antimicrobial sequence motif. The activity was enhanced when the parasin-like N-terminal sequence was also present, as the fragment HIP(1-36) which consists of residues 1-36 in hipposin was more potent than HIP(16-36). Extending HIP(1-36) with three C-terminal residues-thereby constructing the buforin I-like peptide HIP(1-39) (differs from buforin I in only three residues)-increased the activity further. Also, the presence of the C-terminal part of hipposin (residues 40-51) increased the activity, as hipposin was clearly the most potent of all the peptides that were tested. Circular dichroism structural analysis of the peptides revealed that they were all non-structured in aqueous solution. However, trifluoroethanol and the membrane-mimicking entities dodecylphosphocholine micelles and negatively charged liposomes induced (amphiphilic) alpha-helical structuring in hipposin. Judging from the structuring of the individual fragments, the tendency for alpha-helical structuring appeared to be greater in the C-terminal and the buforin II-like middle region of hipposin than in the parasin-like N-terminal region.     

Insights into buforin II membrane translocation from molecular dynamics simulations

Buforin II is a histone-derived antimicrobial peptide that readily translocates across lipid membranes without causing significant membrane permeabilization. Previous studies showed that mutating the sole proline of buforin II dramatically decreases its translocation. As well, researchers have proposed that the peptide crosses membranes in a cooperative manner by forming transient toroidal pores. This paper reports molecular dynamics simulations designed to investigate the structure of buforin II upon membrane entry and evaluate whether the peptide is able to form toroidal pore structures. These simulations showed a relationship between protein–lipid interactions and increased structural deformations of the buforin N-terminal region promoted by proline. Moreover, simulations with multiple peptides show how buforin II can embed deeply into membranes and potentially form toroidal pores. Together, these simulations provide structural insight into the translocation process for buforin II in addition to providing more general insight into the role proline can play in antimicrobial peptides.     

Effect of lipid composition on buforin II structure and membrane entry

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.     

Buforins: Histone H2A-derived antimicrobial peptides from toad stomach

Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.     

Effect of proline position on the antimicrobial mechanism of buforin II

Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.     

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.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 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.     

Importance of the disulfide bridges in the antibacterial activity of human hepcidin

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.     

Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria

The widespread natural sources‐derived cationic peptides have been reported to reveal bacterial killing and/or growth‐inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug‐resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib‐Arg‐Aib‐Ala sequences, showed strong antibacterial activity against both Gram‐negative and Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug‐resistant Pseudomonas aeruginosa, known as proteases‐secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib‐derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Structural contributions to the intracellular targeting strategies of antimicrobial peptides

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide-DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic α-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an α-helix structure is not required for the DNA binding and condensation activity of buforin II amide.     

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.     

Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.     

Solution NMR studies of amphibian antimicrobial peptides: Linking structure to function?

The high-resolution three-dimensional structure of an antimicrobial peptide has implications for the mechanism of its antimicrobial activity, as the conformation of the peptide provides insights into the intermolecular interactions that govern the binding to its biological target. For many cationic antimicrobial peptides the negatively charged membranes surrounding the bacterial cell appear to be a main target. In contrast to what has been found for other classes of antimicrobial peptides, solution NMR studies have revealed that in spite of the wide diversity in the amino acid sequences of amphibian antimicrobial peptides (AAMPs), they all adopt amphipathic α-helical structures in the presence of membrane-mimetic micelles, bicelles or organic solvent mixtures. In some cases the amphipathic AAMP structures are directly membrane-perturbing (e.g. magainin, aurein and the rana-box peptides), in other instances the peptide spontaneously passes through the membrane and acts on intracellular targets (e.g. buforin). Armed with a high-resolution structure, it is possible to relate the peptide structure to other relevant biophysical and biological data to elucidate a mechanism of action. While many linear AAMPs have significant antimicrobial activity of their own, mixtures of peptides sometimes have vastly improved antibiotic effects. Thus, synergy among antimicrobial peptides is an avenue of research that has recently attracted considerable attention. While synergistic relationships between AAMPs are well described, it is becoming increasingly evident that analyzing the intermolecular interactions between these peptides will be essential for understanding the increased antimicrobial effect. NMR structure determination of hybrid peptides composed of known antimicrobial peptides can shed light on these intricate synergistic relationships. In this work, we present the first NMR solution structure of a hybrid peptide composed of magainin 2 and PGLa bound to SDS and DPC micelles. The hybrid peptide adopts a largely helical conformation and some information regarding the inter-helix organization of this molecule is reported. The solution structure of the micelle associated MG2-PGLa hybrid peptide highlights the importance of examining structural contributions to the synergistic relationships but it also demonstrates the limitations in the resolution of the currently used solution NMR techniques for probing such interactions. Future studies of antimicrobial peptide synergy will likely require stable isotope-labeling strategies, similar to those used in NMR studies of proteins.     

In vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes.

Antibacterial peptides have been isolated from a wide range of species. Some of these peptides act on microbial membranes, disrupting their barrier function. With the increasing development of antibiotic resistance by bacteria, these antibacterial peptides, which have a new mode of action, have attracted interest as antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides that act selectively on microbial membranes. In vitro display techniques are powerful tools to select biologically functional peptides from peptide libraries. Here, we used the ribosome display system to form peptide-ribosome-mRNA complexes in vitro from nucleotides encoding a peptide library, as well as immobilized model membranes, to select specific sequences that recognize bacterial membranes. This combination of ribosome display and immobilized model membranes was effective as an in vitro high-throughput screening system and enabled us to identify motif sequences (ALR, KVL) that selectively recognized the bacterial membrane. Owing to host toxicity, it was not possible to enrich any sequence expected to show antimicrobial activity using another in vitro system, e.g. phage display. The synthetic peptides designed from these enriched motifs acted selectively on the bacterial model membrane and showed antibacterial activity. Moreover, the motif sequence conferred selectivity onto native peptides lacking selectivity, and decreased mammalian cell toxicity of native peptides without decreasing their antibacterial activity.     

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.     

Interactions of the novel antimicrobial peptide buforin 2 with lipid bilayers: proline as a translocation promoting factor

Buforin 2 is an antimicrobial peptide discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans. The 21-residue peptide with +6 net charge shows antimicrobial activity an order of magnitude higher than that of magainin 2, a membrane-permeabilizing antimicrobial peptide from Xenopus laevis [Park, C. B., Kim, M. S., and Kim, S. C. (1996) Biochem. Biophys. Res. Commun. 218, 408-413]. In this study, we investigated the interactions of buforin 2 with phospholipid bilayers in comparison with magainin 2 to obtain insight into the mechanism of action of buforin 2. Equipotent Trp-substituted peptides were used to fluorometrically monitor peptide-lipid interactions. Circular dichroism measurements showed that buforin 2 selectively bound to liposomes composed of acidic phospholipids, assuming a secondary structure similar to that in trifluoroethanol/water, which is an amphipathic helix distorted around Pro(11) with a flexible N-terminal region [Yi, G. S., Park, C. B., Kim, S. C., and Cheong, C. (1996) FEBS Lett. 398, 87-90]. Magainin 2 induced the leakage of a fluorescent dye entrapped within lipid vesicles coupled to lipid flip-flop. These results have been interpreted as the formation of a peptide-lipid supramolecular complex pore [Matsuzaki, K. (1998) Biochim. Biophys. Acta 1376, 391-400]. Buforin 2 exhibited much weaker membrane permeabilization activity despite its higher antimicrobial activity. In contrast, buforin 2 was more efficiently translocated across lipid bilayers than magainin 2. These results suggested that the ultimate target of buforin 2 is not the membrane but intracellular components. Furthermore, buforin 2 induced no lipid flip-flop, indicating that the mechanism of translocation of buforin 2 is different from that of magainin 2. The role of Pro was investigated by use of a P11A derivative of buforin 2. The derivation caused a change to magainin 2-like secondary structure and membrane behavior. Pro(11) was found to be a very important structural factor for the unique properties of buforin 2.