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

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

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8–Cys23 and Cys11–Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.     

The introduction of l-phenylalanine into antimicrobial peptide protonectin enhances the selective antibacterial activity of its derivative phe-Prt against Gram-positive bacteria

Protonectin was a typical amphiphilic antimicrobial peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria. In the present study, when its eleventh amino acid in the sequence was substituted by phenylalanine, the analog named phe-Prt showed potent antimicrobial activity against Gram-positive bacteria, but no antimicrobial activity against Gram-negative bacteria, indicating a significant selectivity between Gram-positive bacteria and Gram-negative bacteria. However, when Gram-negative bacteria were incubated with EDTA, the bacteria were susceptible to phe-Prt. Next, the binding effect of phe-Prt with LPS was determined. Our result showed that LPS could hamper the bactericidal activity of phe-Prt against Gram-positive bacteria. The result of zeta potential assay further confirmed the binding effect of phe-Prt with LPS for it could neutralize the surface charge of E. coli and LPS. Then, the effect of phe-Prt on the integrity of outer membrane of Gram-negative bacteria was determined. Our results showed that phe-Prt had a much weaker disturbance to the outer membrane of Gram-negative bacteria than the parent peptide protonectin. In summary, the introduction of l-phenylalanine into the sequence of antimicrobial peptide protonectin made phe-Prt show significant selectivity against Gram-positive bacteria, which could partly be attributed to the delay effect of LPS for phe-Prt to access to cell membrane. Although further study is still needed to clarify the exact mechanism of selectivity, the present study provided a strategy to develop antimicrobial peptides with selectivity toward Gram-positive and Gram-negative bacteria.

     

Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic ��-Helical Peptides

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.     

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks.     

Solution structure and model membrane interactions of temporins-SH, antimicrobial peptides from amphibian skin. A NMR spectroscopy and differential scanning calorimetry study

Temporin-SHa and temporin-SHc are 13 residue long antimicrobial peptides from frog skin that have similar sequences but differ markedly in their membrane-damaging properties. Temporin-SHa contains a single basic lysine residue and has a unique antimicrobial spectrum of action among temporins, being very potent against Gram-positive and Gram-negative bacteria, yeasts, fungi, and protozoa. Temporin-SHc, which contains a single basic histidine residue, is inactive against Gram-negative bacteria, has a reduced efficacy against Gram-positive bacteria, but is still active against yeasts and fungi. Temporin-SHb, with no basic residue, has no antimicrobial activity. The three-dimensional structures of the peptides bound to SDS micelles were analyzed by CD and NMR spectroscopy combined with restrained molecular dynamics calculations. The peptides adopt well-defined amphipathic alpha-helical structures extending from residue 3 to residue 12, when bound to SDS micelles. The structures are stabilized by extensive interactions between aliphatic and aromatic side chains on the nonpolar face. Relaxation enhancements caused by paramagnetic probes showed that the peptides adopt nearly parallel orientations to the micelle surface and do not deeply penetrate into the micelle. The interaction of the peptides with model membranes was investigated by differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles and membrane-permeabilization assays on calcein-loaded large unilamellar vesicles. Calorimetric data indicated that both temporin-SHa and -SHc reside at the hydrocarbon core-water interface of the anionic lipid bilayer but interact with anionic bilayers in a very different manner. This suggests that the charge-induced activity of temporins-SH for bacterial cells is due to changes in the membrane-disturbing mechanism of the bound peptides.     

Synthesis,biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH₂), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100‑alanyl‑hexadecyl‑1‑amine (BP100-Ala-NH-C₁₆H₃₃) and cyclo(1‑4)‑d‑Cys¹, Ile², Leu³, Cys⁴-BP100 (Cyclo(1‑4)‑cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C₁₆H₃₃ acquired α-helical conformation, while Cyclo(1‑4)‑cILC-BP100) was partly α-helical and partly β-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.     

In Silico Design,Synthesis, and In Vitro Evaluation of Novel Amphipathic Short Linear Peptides Against Clinically Relevant Bacterial Biofilms

Microbial biofilms are organized communities of cells that are associated with a wide spectrum of resistant and chronic infections that lead to the treatment failure. Accordingly, there is an urgent demand to create novel effective therapeutic drugs that can inhibit biofilm formation with new mechanisms of action to surmount the current escalating resistance. In this study, in silico hybrid model was utilized to develop three novel short linear peptides (4, 5, and 6) with potential biofilm inhibiting activities (scores?>?1.0). The peptides were composed of cationic and hydrophobic residues. They were synthesized using solid-phase strategy. Synthesized peptides were purified and characterized by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization spectroscopy, respectively. They were evaluated using in vitro assay as potential inhibitors of clinically relevant Gram-positive and Gram-negative biofilms. Peptide (4) with five positive charges at physiological pH (4 cationic moieties and W:R?=?1:4) showed activity against biofilms of Gram-positive strains (Staphylococcus epidermidis and Listeria monocytogenes). On the other hand, peptide (5) with six positive charges (5 cationic moieties and W:R?=?2:2) demonstrated activity against Gram-positive (S. epidermidis) and Gram-negative (Escherichia coli) biofilms. Interestingly, peptide (6), with seven positive charges (6 cationic moieties and W:R?=?2:5) revealed higher and broader spectrum of activity against biofilms of Gram-positive (S. epidermidis, S. aureus, L. monocytogenes) and Gram-negative (E. coli).

     

Molecular cloning of a cDNA encoding atypical antimicrobial and cytotoxic Brevinin-1Ja from the skin of the Japanese brown frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).     

Synthesis,micellisation and interaction of novel quaternary ammonium compounds derived from l-Phenylalanine with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as model membrane in relation to their antibacterial activity,and their selectivity over human red blood cells

A series of quaternary ammonium compounds (QUATS) derived from l-Phenylalanine have been synthesized and their antibacterial efficiencies were determined against various strains of Gram-positive and Gram-negative bacteria. The antibacterial activity increased with increasing chain length, exhibiting a cut-off effect at C₁₄ for Gram-positive and C₁₂ for Gram-negative bacteria. The l-Phenylalanine QUATS displayed enhanced antibacterial properties with a higher cut-off point compared to their corresponding l-Phenylalanine ester hydrochlorides. The CMC was correlated with the MIC, inferring that micellar activity contributes to the cut-off effect in antibacterial activity. The hemolytic activities (HC₅₀) of the QUATS against human red blood cells were also determined to illustrate the selectivity of these QUATS for bacterial over mammalian cells. In general, the MIC was lower than the HC₅₀, and assessment of the micellar contribution to the antibacterial and hemolytic evaluation in TBS as a common medium confirmed that these QUATS can act as antibacterial, yet non-toxic molecules at their monomer concentrations. The interaction of the QUATS with the phospholipid vesicles (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) in the presence of 1-anilino-8-naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probes showed that the presence of the quaternary ammonium moiety causes an increase in hydrophobic interactions, thus causing an increase in antibacterial activity.     

Temporin-SHf,a New Type of Phe-rich and Hydrophobic Ultrashort Antimicrobial Peptide

Because issues of cost and bioavailability have hampered the development of gene-encoded antimicrobial peptides to combat infectious diseases, short linear peptides with high microbial cell selectivity have been recently considered as antibiotic substitutes. A new type of short antimicrobial peptide, designated temporin-SHf, was isolated and cloned from the skin of the frog Pelophylax saharica. Temporin-SHf has a highly hydrophobic sequence (FFFLSRIFa) and possesses the highest percentage of Phe residues of any known peptide or protein. Moreover, it is the smallest natural linear antimicrobial peptide found to date, with only eight residues. Despite its small size and hydrophobicity, temporin-SHf has broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria and yeasts, with no hemolytic activity. CD and NMR spectroscopy combined with restrained molecular dynamics calculations showed that the peptide adopts a well defined non-amphipathic α-helical structure from residue 3 to 8, when bound to zwitterionic dodecyl phosphocholine or anionic SDS micelles. Relaxation enhancement caused by paramagnetic probes showed that the peptide adopts nearly parallel orientations to the micelle surface and that the helical structure is stabilized by a compact hydrophobic core on one face that penetrates into the micelle interior. Differential scanning calorimetry on multilamellar vesicles combined with membrane permeabilization assays on bacterial cells indicated that temporin-SHf disrupts the acyl chain packing of anionic lipid bilayers, thereby triggering local cracks and microbial membrane disintegration through a detergent-like effect probably via the carpet mechanism. The short length, compositional simplicity, and broad-spectrum activity of temporin-SHf make it an attractive candidate to develop new antibiotic agents.     

Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negative bacteria by fusion with LPS-targeting peptide

Antimicrobial peptides (AMPs) provide a potential source of new antimicrobial therapeutics for the treatment of multidrug-resistant pathogens. To develop Gram-negative selective AMPs that can inhibit the effects of lipopolysaccharide (LPS)-induced sepsis, we added various rationally designed LPS-targeting peptides [amino acids 28–34 of lactoferrin (Lf28–34), amino acids 84–99 of bactericidal/permeability increasing protein (BPI84–99), and de novo peptide (Syn)] to the potent AMP, GNU7 (RLLRPLLQLLKQKLR). Compared to our original starting peptide GNU7, hybrid peptides had an 8- to 32-fold improvement in antimicrobial activity against Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium. Among them, Syn-GNU7 showed the strongest LPS-binding and -neutralizing activities, thus allowing it to selectively eliminate Gram-negative bacteria from within mixed cultures. Our results suggest that LPS-targeting peptides would be useful to increase the antimicrobial activity and selectivity of other AMPs against Gram-negative bacteria.     

Trichoplaxin — A new membrane-active antimicrobial peptide from placozoan cDNA

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.     

Studies of synthetic peptide analogs of the amphipathic helix. Correlation of structure with function

The four peptide analogs of the amphipathic helix whose interactions with dimyristoyl phosphatidylcholine were described in the preceding paper were compared with apolipoproteins (apo) A-I and A-II in ability to displace native apolipoprotein from high density lipoprotein (HDL) and in ability to activate lecithin:cholesterol acyltransferase. The rank order of the ability of the four peptide analogs to displace apo-A-I from intact HDL was 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, the same order suggested in the preceding paper for relative lipid affinities. Modified HDL from which 40% of the apo-A-I had been displaced by 18A was indistinguishable from unmodified HDL in its ability to act as a lecithin:cholesterol acyltransferase substrate. This suggests that the easily displaced apo-A-I molecules in polydisperse HDL are relatively ineffectual as lecithin:cholesterol acyltransferase activators and/or 18A replaces the lecithin:cholesterol acyltransferase activity lost. The peptide analog 18A-Pro-18A was found to be a powerful activator of lecithin:cholesterol acyltransferase when incubated with unilamellar egg phosphatidylcholine (PC) vesicles, reaching 140% of the activity of apo-A-I at a 1:1.75 peptide-to-egg PC ratio. In another experiment, it was found that discoidal egg PC complexes of 18A-Pro-18A, 18A, and des-Val10-18A, formed by cholate dialysis, had 30-45% of the activity of apo-A-I/egg PC discoidal complexes, also formed by cholate dialysis, at the same peptide/lipid weight ratio. Examination of the structures formed when the 18A-Pro-18A peptide was incubated with unilamellar egg PC vesicles indicated that the ability of 18A-Pro-18A to exceed apo-A-I in lecithin:cholesterol acyltransferase activating ability is due to the spontaneous conversion by 18A-Pro-18A of egg PC vesicles to small protein annulus-bilayer disc structures. Apo-A-I, apo-A-II, nor any of the other three peptide analogs of the amphipathic helix studied were able to convert a significant fraction of egg PC unilamellar vesicles to discoidal structures.     

Liposomal Hexadecylphosphocholine: Characterization and Effects on Adherent Tumor Cells

Abstract

We describe the preparation of small unilamellar and multilamellar vesicles from hexadecylphosphocholine, cholesterol and 1,2-dipalmitoyl-sn-glycero-phosphoglycerol in the molar ratio 4/5/1. Particle size and chemical stability of two types of liposomes, small unilamellar vesicles and lyophilized, freshly resuspended multilamellar vesicles were proved to be stable for at least 12 months. Compared to hexadecylphosphocholine in free form, liposomal hexadecylphosphocholine showed remarkably reduced hemolysis which did not change during storage. Fluorescence microscopy showed the uptake of propidium iodide containing hexadecylphosphocholine liposomes by KB and MDA-MB 231 tumor cells. Free propidium iodide was not incorporated into these cells. Although cytotoxicity seemed to be reduced in liposomal preparations, hexadecylphosphocholine liposomes still affected cultured tumor cells to a great extent. In relatively low concentrations they induced shape alteration, smoothing of the cell surface and blebbing.     

The synthetic antimicrobial peptide IKR18 displays anti-infectious properties in Galleria mellonella in vivo model

Antimicrobial peptides (AMPs) are promising tools for developing new antibiotics. We described the design of IKR18, an AMP designed with the aid of computational tools. IKR18 showed antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). CD studies revealed that IKR18 assumes an alpha-helical structure in the membrane-mimetic environment. The action mechanism IKR18 involves damage to the bacteria membrane, as demonstrated by Sytox green uptake. Furthermore, IKR18 displayed synergic and additive effects in combination with antibiotics ciprofloxacin and vancomycin. The peptide showed anti-biofilm activity in concentration and efficiency compared with commercial antibiotics, involving the direct death of bacteria, as confirmed by scanning electron microscopy. The anti-infective activity of IKR18 was demonstrated in the Galleria mellonella model infected with S. aureus, MRSA, and Acinetobacter baumannii. The novel bioinspired peptide, IKR18, proved to be effective in the control of bacterial infection, opening opportunities for the development of further assays, including preclinical models.     

Scolopendin 2, a cationic antimicrobial peptide from centipede,and its membrane-active mechanism

Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3′-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane.     

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

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

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.