Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity of peptides derived from human ß‐amyloid precursor protein

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin‐binding disulfide‐constrained loop region of human ß‐amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C‐terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL‐37, these peptides efficiently killed the Gram‐negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram‐positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop‐structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

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.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Design of antimicrobial peptide arenicin analogs with improved therapeutic indices

β‐Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β‐hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template‐based approach was used to create therapeutically valuable analogs of arenicin‐1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin‐1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non‐polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50‐fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin‐1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin‐1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of short cationic antimicrobial peptidomimetics containing arginine analogues

Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1–8) containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram‐positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram‐negative (Escherichia coli and Klebsiella pneumoniae) bacterial strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives (MIC values range 7.5–50 µg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable activity against the tested Gram‐negative bacteria and C. albicans. The most active compounds (1–2 and 5–6) have been tested against Gram‐positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these compounds were never up to 200 µg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable loss of the antibacterial action (compounds 3–4 and 7–8) against both Gram‐positive and Gram‐negative bacterial strains. Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for compound 5. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library

We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram‐negative and ‐positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1‐h incubation at a concentration of 62.5 µM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt‐dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13‐treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced α‐helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library. Copyright © 2009 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.     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

Cell selectivity and interaction with model membranes of Val/Arg‐rich peptides

Antimicrobial peptides are major components of the innate self‐defence system and a large number of peptides have been designed to study the mechanism of action. In the present study, a small combinatorial library was designed to study whether the biological activity of Val/Arg‐rich peptides is associated with targeted cell membranes. The peptides were produced by segregating hydrophilic residues on the polar side and hydrophobic residues on the opposite side. The peptides displayed strong antimicrobial activity against Gram‐negative and Gram‐positive bacteria, but weak haemolysis even at a concentration of 256 µM. CD spectra showed that the peptides formed α‐helical‐rich structure in the presence of negatively charged membranes. The tryptophan fluorescence and quenching experiments indicated that the peptides bound preferentially to negatively charged phospholipids over zwitterionic phospholipids, which corresponds well with the biological activity data. In the in vivo experiment, the peptide G6 decreased the bacterial counts in the mouse peritoneum and increased survival after 7 days. Overall, a high binding affinity with negatively charged phospholipids correlated closely with the cell selectivity of the peptides and some peptides in this study may be likely candidates for the development of antibacterial agents. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial peptides derived from different animals: comparative studies of antimicrobial properties,cytotoxicity and mechanism of action

Animals posses a large variety of antimicrobial peptides (AMPs) that serve as effective components in innate host defenses against microbial infections. These antimicrobial peptides differ in amino acid composition, range of antimicrobial specificities, hemolysis, cytotoxicity and mechanisms of action. This study was designed to evaluate their therapeutic potential of the following six antimicrobial peptides initially found from animals: cecropin P1, indolicidin, LL-37, palustrin-OG1, LFP-20 and LFB-11. Our results indicated that cecropin P1 possessed the most desired biological activity, with fast and potent antimicrobial activity but only slight hemolytic or cytotoxic activity against human cells. Indolicidin was more effective against gram-positive bacteria but with higher hemolytic and cytotoxic activity on human peripheral blood mononuclear cell (PBMCs) (P < 0.05). Although LFP-20 and LFB-11 had moderate activity against tested strains and need 30 min to kill E. coli, they showed almost no hemolytic and cytotoxic activity towards PBMCs (P < 0.01). Indolicidin could form pores of well-defined structure in bacterial membranes whereas lysis of E. coli cells was observed after addition LFB-11 and LL-37 at 1 × MIC for 1 h. LL-37 treatment could lead to the leakage of entire bacterial cytoplasmic contents. The most obvious phenomenon was protuberant structures on the E. coli cell surface after incubation with LFP-20, cecropin P1 and palustrin-OG1. The results presented here illustrate that AMPs derived from different animals exhibited different antimicrobial characteristics. Because of their potent and broad-spectrum antimicrobial activity, low cytotoxicity towards normal cells, and the unique mechanism of action, these peptides may provide the impetus for the development of novel strategies for the prevention of bacterial infections in animals.     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Dependence of prePhoA-phospholipid interaction in vivo and in vitro on charge of signal peptide N-terminus and content of anionic phospholipids in membranes

Replacement of the positively charged signal peptide with neutral or negatively charged peptides due to substitution of Lys(–20) in the N-terminal region of the signal peptide leads to decreases in the rate of prePhoA membrane translocation in vivo and in the efficiency of prePhoA insertion into liposomes in vitro. The effect of anionic phospholipids on prePhoA insertion into model membranes is determined by the signal peptide N-terminus charge, while the dependence of prePhoA translocation across the cytoplasmic membrane in vivo is not, under the studied variations in the content of anionic phospholipids. This is evidence of the possibility of direct electrostatic interaction between the signal peptide N-terminus and anionic phospholipids, which in vivo, however, seems to involve some proteins of the Sec machinery.     

Synthetic analogs of anoplin show improved antimicrobial activities

We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin‐resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin‐resistant Enterococcus faecium (ATCC 700221) (VRE), and Candida albicans (ATCC 200955). The anoplin analogs contain substitutions in amino acid positions 2, 3, 5, 6, 8, 9, and 10. We use these peptides to study the effect of altering the charge and hydrophobicity of anoplin on activity against red blood cells and microorganisms. We find that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few compounds are more specific than anoplin. Both 2Nal⁶ and Cha⁶ show improved therapeutic index against all strains tested. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Structure‐activity relationship of Trp‐containing analogs of the antimicrobial peptide gomesin

Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. In this study, we analyzed three Gm analogs, [Trp¹]‐Gm, [Trp⁷]‐Gm, and [Trp⁹]‐Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. The incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (~8 mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0–15 mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3‐6.0 mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent‐peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally‐restricted in the presence of SDS, probably because this residue is located at the N‐terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.     

Selective cytotoxicity following Arg-to-Lys substitution in tritrpticin adopting a unique amphipathic turn structure

In antimicrobial peptides, the cationic property due to basic amino acids has been widely recognized as an important factor to promote electrostatic interaction with negatively charged phospholipids. However, little is known about the differences between two basic residues, Arg and Lys, in membrane binding affinity. Tritrpticin is an Arg- or Trp-rich antimicrobial peptide with a broad spectrum of antibacterial and antifungal activity. To investigate the structural and functional differences between Arg and Lys residues, here we designed and synthesized Arg-containing peptides, tritrpticin and SYM11, and their counterpart Lys-substituted peptides, TRK and SYM11KK, respectively. Although there were no remarkable conformational differences between Arg-containing and Lys-substituted peptides, TRK and SYM11KK exhibited almost two-fold enhanced antibacterial activity but significantly reduced hemolytic activity as compared to tritrpticin and SYM11, respectively. Furthermore, Arg-containing peptides showed strong binding affinity to both zwitterionic and anionic liposomes, whereas Lys-substituted peptides interacted weakly with zwitterionic liposomes but strongly with anionic liposomes. These results suggest that the primary amine of Lys interacts less electrostatically with zwitterionic phospholipids than the guanidinium group of Arg. Our results obtained in this study may be helpful in the design of drugs that target negatively charged phospholipids.