magainin-2-peptide--xenopus and Hemolysis

Antimicrobial peptides (AMPs) are promising therapeutic agents for treating antibiotic-resistant bacterial infections. Previous studies showed that magainin 2 (isolated from African clawed fogs

Topics: Acinetobacter baumannii; Animals; Anti-Bacterial Agents; Biofilms; Cell Line; Cell Survival; Circular Dichroism; Drug Resistance, Multiple, Bacterial; Erythrocytes; Hemolysis; Humans; Magainins; Mice; Xenopus Proteins

Antimicrobial peptides (AMPs) are significant components of the innate immune system and play indispensable roles in the resistance to bacterial infection. Here, we investigated the antimicrobial activity of lycosin-I, a 24-residue cationic anticancer peptide derived from Lycosa singorensis with high structural similarity to several cationic and amphiphilic antimicrobial peptides. The antimicrobial activity of lycosin-I against 27 strains of microbes including bacteria and fungi was examined and compared with that of the Xenopus-derived AMP magainin 2 using a microdilution assay. Lycosin-I inhibited the growth of most microorganisms at low micromolar concentrations, and was a more potent inhibitor than magainin 2. Lycosin-I showed rapid, selective and broad-spectrum bactericidal activity and a synergistic effect with traditional antibiotics. In vivo, it showed potent bactericidal activity in a mouse thigh infection model. High Mg2+ concentrations reduced the antibacterial effect of lycosin-I, implying that the peptide might directly interact with the bacterial cell membrane. Uptake of the fluorogenic dye SYTOX and changes in the surface of lycosin-Itreated bacterial cells observed by scanning electron microscopy confirmed that lycosin-I permeabilized the cell membrane, resulting in the rapid bactericidal effect. Taken together, our findings indicate that lycosin-I is a promising peptide with the potential for the development of novel antibacterial agents.

Topics: Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Erythrocytes; Hemolysis; Humans; Magainins; Magnesium; Mice; Microbial Sensitivity Tests; Microbial Viability; Spider Venoms; Spiders; Staphylococcal Infections; Xenopus Proteins

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacillus anthracis; Burkholderia cepacia; Cathelicidins; Erythrocytes; Francisella tularensis; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Magainins; Microbial Sensitivity Tests; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Protein Structure, Secondary; Recombinant Fusion Proteins; Xenopus Proteins; Yersinia pseudotuberculosis

The antimicrobial peptides magainin 2 and PGLa isolated from the skin of the African clawed frog Xenopus laevis show marked functional synergism. We have proposed that the two peptides form a heterodimer composed of parallel helices with strong membrane permeabilizing activity [Hara, T., Mitani, Y., Tanaka, K., Uematsu, N., Takakura, A., Tachi, T., Kodama, H., Kondo, M., Mori, H., Otaka, A., Fujii, N., and Matsuzaki, K. (2001) Biochemistry 40, 12395-12399]. In this study, to elucidate the molecular mechanism of the synergy, we synthesized a chemically fixed heterodimer and investigated in detail the interaction of the hybrid peptide with bacteria, erythrocytes, and lipid bilayers. The hybrid peptide showed antimicrobial activity and membrane permeabilizing activity against negatively charged membranes, similar to or even stronger than those of a physical equimolar mixture of magainin and PGLa, indicating that the synergy is due to the formation of a parallel heterodimer. The heterodimer assumed a more oblique orientation than the component peptides. In contrast, the cross-linking of the two peptides significantly strengthened the action against erythrocytes and zwitterionic lipid bilayers by enhancing the affinity for membranes without changing the basic mode of action. Thus, the separate production of mutually recognizing peptides without cross-linking appears to be a good way to increase selective toxicity.

Topics: Adult; Amino Acid Sequence; Antimicrobial Cationic Peptides; Cell Membrane Permeability; Dimerization; Drug Synergism; Escherichia coli; Hemolysis; Humans; Magainins; Male; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Protein Precursors; Spectroscopy, Fourier Transform Infrared; Staphylococcus epidermidis; Unilamellar Liposomes; Xenopus Proteins

Antimicrobial peptides (AMPs) are a diverse group of proteinaceous compounds ranging in size, complexity and antimicrobial spectrum. The activity of AMPs against gut pathogens warrants the study of the interaction of AMPs with the mammalian gastrointestinal tract. In particular, the investigation of the in vitro cytotoxicity of these peptides is critical before they can be considered in clinical infections. The cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin was investigated in two gastrointestinal cell models (HT29 and Caco-2) with the MTT conversion assay, neutral red dye uptake assay and compared with that of vancomycin. The hemolytic activities were also investigated in sheep erythrocytes and the effect of AMPs on paracellular permeability was examined by transepithelial resistance (TEER) and TEM. Gallidermin was the least cytotoxic AMP followed by nisin A, magainin I, magainin II and melittin. Melittin and nisin were the only peptides to result in significant hemolysis. However, while nisin caused hemolysis at concentrations which were 1000-fold higher than those required for antimicrobial activity, melittin was hemolytic at concentrations in the same order of magnitude as its antimicrobial activity. Melittin was the only AMP to affect paracellular permeability. Long term melittin treatment also resulted in loss of microvilli, an increase in cell debris and destruction of intestinal tight junctions and cell-cell adhesion. Gallidermin shows most promise as a therapeutic agent, with relatively low cytotoxicity and potent antimicrobial activities. Melittin, while showing little potential as an antimicrobial agent, may have potential in delivery of poorly bioavailable drugs.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteriocins; Caco-2 Cells; Cell Membrane; Cell Survival; Epithelial Cells; Erythrocytes; Escherichia coli; Hemolysis; HT29 Cells; Humans; Intestines; L-Lactate Dehydrogenase; Magainins; Melitten; Micrococcus luteus; Microscopy, Electron, Transmission; Nisin; Peptides; Sheep; Vancomycin; Xenopus Proteins

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Base Sequence; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Fungi; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Magainins; Mice; Molecular Sequence Data; Pichia; Recombinant Fusion Proteins; Spectrometry, Mass, Electrospray Ionization; Xenopus Proteins

The emergence of membrane-active antimicrobial peptides as new alternatives against pathogens with multiantibiotic resistance requires the design of better analogues. Among the different physicochemical parameters involved in the optimization of linear antimicrobial peptides, positional hydrophobicity has recently been incorporated. This takes into consideration the concept of the topological distribution of hydrophobic residues throughout the sequence rather than the classical concept of hydrophobicity as a global parameter of the peptide, calculated as the summation of the individual hydrophobicities of the residues. In order to assess the contribution of this parameter to the leishmanicidal mechanisms of magainin 2 analogues, the activities of two of these analogues, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), which have similar charges, amino acid compositions, and hydrophobicities but different positional hydrophobicities, against Leishmania donovani promastigotes were assayed (T. Tachi, R. F. Epand, R. M. Epand, and K. Matsuzaki, Biochemistry 41:10723-10731, 2002). The activities were compared with that of the parental peptide, F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS). The three peptides were active at micromolar concentrations, in the order MG-H2 > MG-H1 > F5W-magainin 2. These activities differ from their hemolytic and bactericidal activities. The results demonstrate that positional hydrophobicity, which reflects the presence of short stretches of sequences rich in hydrophobic amino acids, plays an important role in the activities of leishmanicidal peptides.

Topics: Adenosine Triphosphate; Animals; Antimicrobial Cationic Peptides; Antiprotozoal Agents; Cell Membrane; Cell Membrane Permeability; Chemical Phenomena; Chemistry, Physical; Energy Metabolism; Fluorometry; Glycocalyx; Hemolysis; Indicators and Reagents; Leishmania donovani; Magainins; Membrane Potentials; Microscopy, Electron; Peptides; Structure-Activity Relationship; Xenopus laevis; Xenopus Proteins

A cysteine substitution analogue of magainin-2 amide (magainin-F12W, N22C; denoted here as mag-N22C), and a disulfide-linked dimer prepared by air oxidation [(mag-N22C)(2)], were compared in their ability to release carboxyfluorescein (CF) from 100-nm large unilamellar vesicles (LUV) and to kill the Gram negative bacteria Stenotrophomonas maltophilia and Escherichia coli. The disulfide-dimerized peptide showed enhanced permeabilization and antimicrobial activity, when compared with the monomeric peptide, that was particularly marked at very low peptide concentrations. The enhanced CF-releasing activity of the dimer at low concentrations in vesicles results from (i) enhanced binding to negatively charged membrane surfaces and (ii) a low concentration dependence for permeabilization in the dimer compared to the monomer. The unique properties of the dimeric peptide suggest a role for structural diversity of antimicrobial peptides in frog skin, including the recent identification of a heterodimer composed of disulfide-linked amphipathic helical peptides [Batista et al. (2001) FEBS Lett. 494, 85-89]. Disulfide-dimerization of pore-forming, positively charged, amphipathic helical peptides may be a useful general approach to the generation of peptide antimicrobials having activity at very low concentrations.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Membrane Permeability; Circular Dichroism; Dimerization; Disulfides; Erythrocytes; Escherichia coli; Fluoresceins; Hemolysis; Humans; Lipid Bilayers; Magainins; Molecular Sequence Data; Protein Binding; Spectrometry, Fluorescence; Stenotrophomonas maltophilia; Structure-Activity Relationship; Xenopus laevis; Xenopus Proteins

We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Chromatography, High Pressure Liquid; Circular Dichroism; Gills; Hemolysis; Humans; Immunoenzyme Techniques; Magainins; Mass Spectrometry; Melitten; Molecular Sequence Data; Protein Conformation; Protein Isoforms; Protein Transport; Sea Bream; Sequence Homology, Amino Acid; Xenopus laevis; Xenopus Proteins

A series of peptoid oligomers were designed as helical, cationic, and facially amphipathic mimics of the magainin-2 amide antibacterial peptide. We used circular dichroism spectroscopy to determine the conformation of these peptoids in aqueous buffer and in the presence of bacterial membrane-mimetic lipid vesicles, composed of a 7:3 mol ratio of POPE:POPG. We found that certain peptoids, which displayed characteristically helical CD in buffer and lipid vesicles, exhibit selective (nonhemolytic) and potent antibacterial activity against both Gram-positive and Gram-negative bacteria. In contrast, peptoids that exhibit weak CD, reminiscent of that of a peptide random coil, were ineffective antibiotics. In a manner similar to the natural magainin peptides, we find a correlation between peptoid lipophilicity and hemolytic propensity. We observe that a minimum length of approximately 12 peptoid residues may be required for antibacterial activity. We also see evidence that a helix length between 24 and 34 A may provide optimal antibacterial efficacy. These results provide the first example of a water-soluble, structured, bioactive peptoid.

Topics: Amides; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biomimetic Materials; Cations; Circular Dichroism; Drug Design; Glycine; Hemolysis; Magainins; Microbial Sensitivity Tests; Protein Structure, Secondary; Xenopus Proteins

Cationic antimicrobial peptides are promising candidates as novel antibiotics of clinical usefulness. Magainin 2, a representative antimicrobial peptide isolated from the skin of the African clawed frog Xenopus leavis, electrostatically recognizes anionic lipids that are abundant in bacterial membranes, forming a peptide-lipid supramolecular complex pore, whereas the peptide does not effectively bind to zwitterionic phospholipids constituting the outer leaflets of mammalian cell membranes because of the low hydrophobicity of the peptide [Matsuzaki, K. (1999) Biochim. Biophys. Acta 1462, 1-10]. In this study, two magainin analogues with enhanced hydrophobicity, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), with identical amino acid compositions were designed and interactions with lipid bilayers and biological activities were examined in comparison with those of MG (GIGKWLHSAKKFGKAFVGEIMNS = F5W-magainin 2). The apparent hydrophobicities and hydrophobic moments of MG-H1 and MG-H2, conventionally calculated assuming that all residues are involved in helix formation, were almost the same. MG-H2 behaved like MG except for greatly enhanced activity against zwitterionic membranes and erythrocytes. In contrast, despite a very similar calculated hydrophobicity, the observed hydrophobicity of MG-H1 was larger than that of MG-H2 because of a tendency toward helix fraying near the termini. Therefore, the physicochemical parameters of only the helical portion should be considered in characterizing peptide-lipid interactions, although this point was overlooked in most studies. Moreover, MG-H1 induced aggregation and/or fusion of negatively charged membranes. Furthermore, the peptide hydrophobicity was found to affect pore formation rate, pore size, and pore stability. These observations demonstrate that the hydrophobicity of the peptide also controls the mode of action and is dependent on the position of the hydrophobic amino acids in the peptide sequence.

Topics: Adult; Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Cell Division; Cell Membrane; Cell Membrane Permeability; Circular Dichroism; Dose-Response Relationship, Drug; Erythrocytes; Escherichia coli; Fluorescence; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Lipid Metabolism; Magainins; Male; Microbial Sensitivity Tests; Molecular Sequence Data; Protein Structure, Secondary; Structure-Activity Relationship; Xenopus laevis; Xenopus Proteins

Our basic understanding of how to combat fungal infections has not kept pace with the recent sharp rise in life-threatening cases found particularly among immuno-compromised individuals. Current investigations for new potential antifungal agents have focused on antimicrobial peptides, which are used as a cell-free defense mechanism in all organisms. Unfortunately, despite their high antibacterial activity, most of them are not active toward fungi, the reason of which is not clear. Here, we present a new approach to modify an antibacterial peptide, a magainin analogue, to display antifungal activity by its conjugation with lipophilic acids. This approach has the advantage of producing well-defined changes in hydrophobicity, secondary structure, and self-association. These modifications were characterized in solution at physiological concentrations using CD spectroscopy, tryptophan fluorescence, and analytical ultracentrifugation. In order of increasing hydrophobicity, the attachment to the magainin-2 analogue of (i) heptanoic acid results in a monomeric, unordered structure, (ii) undecanoic acid yields concentration-dependent oligomers of alpha helices, and (iii) palmitic acid yields concentration-independent alpha-helical monomers, a novel lipopeptide structure, which is resistant to proteolytic digestion. Membrane-lipopeptide interactions and the membrane-bound structures were studied using fluorescence and ATR-FTIR in PC/PE/PI/ergosterol (5/2.5/2.5/1, w/w) SUV, which constitute the major components of Candida albicans bilayers. A direct correlation was found between oligomerization of the lipopeptides in solution and potent antifungal activity. These results provide insight to a new approach of modulating hydrophobicity and self-assembly of antimicrobial peptides in solution, without altering the sequence of the peptidic chain. These studies also provide a general means of developing a new group of lipopeptide candidates as therapeutic agents against fungal infections.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Antifungal Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Cryptococcus neoformans; Endopeptidase K; Energy Transfer; Erythrocyte Membrane; Fatty Acids; Hemolysis; Heptanoic Acids; Humans; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Lipoproteins; Magainins; Membrane Lipids; Membrane Potentials; Molecular Sequence Data; Palmitic Acid; Phospholipids; Protein Structure, Secondary; Solutions; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Trypsin; Tryptophan; Ultracentrifugation; Xenopus laevis; Xenopus Proteins

The amphipathic alpha-helical structure is a common motif found in membrane binding polypeptides including cell lytic peptides, antimicrobial peptides, hormones, and signal sequences. Numerous studies have been undertaken to understand the driving forces for partitioning of amphipathic alpha-helical peptides into membranes, many of them based on the antimicrobial peptide magainin 2 and the non-cell-selective cytolytic peptide melittin, as paradigms. These studies emphasized the role of linearity in their mode of action. Here we synthesized and compared the structure, biological function, and interaction with model membranes of linear and cyclic analogues of these peptides. Cyclization altered the binding of melittin and magainin analogues to phospholipid membranes. However, at similar bound peptide:lipid molar ratios, both linear and cyclic analogues preserved their high potency to permeate membranes. Furthermore, the cyclic analogues preserved approximately 75% of the helical structure of the linear peptides when bound to membranes. Biological activity studies revealed that the cyclic melittin analogue had increased antibacterial activity but decreased hemolytic activity, whereas the cyclic magainin 2 analogue had a marked decrease in both antibacterial and hemolytic activities. The results indicate that the linearity of the peptides is not essential for the disruption of the target phospholipid membrane, but rather provides the means to reach it. In addition, interfering with the coil-helix transition by cyclization, while maintaining the same sequence of hydrophobic and positively charged amino acids, allows a separated evaluation of the hydrophobic and electrostatic contributions to binding of peptides to membranes.

Topics: Acinetobacter calcoaceticus; Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bacillus subtilis; Binding Sites; Circular Dichroism; Drug Resistance, Microbial; Erythrocytes; Escherichia coli; Hemolysis; Humans; Liposomes; Magainins; Melitten; Micrococcus luteus; Molecular Sequence Data; Peptides, Cyclic; Permeability; Protein Structure, Secondary; Tryptophan; Xenopus Proteins

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Binding Sites; Escherichia coli; Hemolysis; Humans; Magainins; Microbial Sensitivity Tests; Peptides; Permeability; Protein Conformation; Xenopus Proteins

Magainin 2 and tachyplesin I (T-SS) are membrane-permeabilizing antimicrobial peptides discovered from frog skin and horseshoe crab hemolymph, respectively. They are classified into different secondary structural classes, i.e., alpha-helix and cyclic beta-sheet, respectively. We found that F5W-magainin 2 (MG2) and T-SS exhibited marked synergistic effects against Gram-negative and Gram-positive bacteria without enhancing hemolytic activity as a measure of toxicity. Dye release experiments using liposomes suggested that the selective synergism is mainly due to anionic phospholipid-specific synergism in membrane permeabilization. Furthermore, the cyclic structure of T-SS was found to be necessary for synergism because a linear analogue of T-SS did not show good synergism with MG2. These novel observations suggested the possibility of the development of cocktail therapeutic regimens using combinations of antimicrobial peptides.

Topics: Adult; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Binding Sites; Cell Membrane; Circular Dichroism; DNA-Binding Proteins; Drug Synergism; Erythrocytes; Female; Fluoresceins; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Horseshoe Crabs; Humans; Magainins; Peptides, Cyclic; Phospholipids; Protein Conformation; Skin; Xenopus laevis; Xenopus Proteins

Small antimicrobial peptides are excellent candidates for inclusion in self-processing proteins that could be used to confer pathogen resistance in transgenic plants. Antimicrobial peptides as small as 22 amino acids in length have been designed to incorporate the residual amino acids left from protein processing by the tobacco etch virus'(TEVs') NIa protease. Also, by minimizing the length of these peptides and the number of highly hydrophobic residues, haemolytic activity was reduced without affecting the peptide's antimicrobial activity.

Topics: Agrobacterium tumefaciens; Amino Acid Sequence; Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Fusarium; Hemolysis; Humans; Magainins; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Plant Diseases; Plants, Genetically Modified; Pseudomonas; Xenopus Proteins

MSI-78 is a peptide analog of naturally occurring magainin 2 isolated from the skin of Xenopus laevis. The peptide is known to have one of the strongest antibacterial activities in magainin 2 analogs against methicillin-resistant Staphylococcus aureus (MRSA). To find novel compounds superior to MSI-78, we have further designed, synthesizing 1,1-di(4-aminobutyl)-6-benzylindane (PM4) and 1,1-dibenzyl-6-(4-aminobutyl) indane (PM5), and tested their inhibitory ability of the growth of S. aureus. In an in vitro assay, PM4 showed the same antibacterial activity against the bacterium as MSI-78, and non-hemolytic activity against human red blood cells (RBCs) at the MIC (minimum inhibitory concentration) value, in contrast to the latter. On the other hand, PM5 showed stronger antibacterial activity than MSI-78, but being still accompanied with hemolysis at the MIC value. Otherwise, stronger decarboxylase activity for oxaloacetate was observed in PM5, rather than magainin 2 analogs or Oxaldie 1 as a control peptide, but not in PM4.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antifungal Agents; Antimicrobial Cationic Peptides; Aspergillus; Carboxy-Lyases; Escherichia coli; Helicobacter pylori; Hemolysis; Humans; Indans; Magainins; Molecular Sequence Data; Oxaloacetic Acid; Peptides; Staphylococcus aureus; Structure-Activity Relationship; Xenopus laevis; Xenopus Proteins

In order to elucidate the structure-antibiotic activity relationship of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides, several truncated peptides and the analogues with amino acid substitutions were synthesized and their antibacterial, antitumor and hemolytic activities of were examined. Cecropin A-magainin 2 hybrid analog, L16-CA(1-8)-MA(1-12) (termed as L-CA-MA in this study: KWKLFKKIGIGKFLHLAKKF-NH2), is known to have potent antibacterial and antitumor activity with less hemolytic activity. We found that the C-terminal region of L-CA-MA is more involved in the alpha-helical structure on cell membrane-like environment than N-terminal one by circular dichroism analysis. Deletion of the Gly-Ile-Gly sequence, the central hinge region of L-CA-MA, produced a considerable reduction in antitumor and hemolytic activity rather than an antibacterial one. The insertion of Pro, Gly-Ile or Gly-Pro in this hinge region of L-CA-MA caused retention of both antibacterial and antitumor activity while causing a significant decrease in hemolytic activity. However, the substitution with Gly-Pro-Gly instead of the Gly-Ile-Gly in CA(1-8)-MA(1-12), CA(1-8)-ME(1-12), CA(1-13)-MA(1-13) and CA(1-13)-ME(1-13) hybrids resulted in a drastic decrease in antibacterial, antitumor and hemolytic activity. The increase of hydrophobicity at position 16 in CA(1-8)-MA(1-12) by substituting Trp or Phe induced a significant increase in hemolytic activity without a considerable change in either antibacterial or antitumor activity. Therefore, these results suggested that the appropriate flexibility in the hinge region of CA-MA and CA-ME hybrid peptides and the appropriate hydrophobicity at position 16 in the hydrophobic region of CA (1-8)-MA(1-12) are important in potent antibacterial and antitumor activity with no hemolytic effect.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Bacteria; Circular Dichroism; Erythrocytes; Hemolysis; Humans; Magainins; Melitten; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptides; Protein Structure, Secondary; Recombinant Fusion Proteins; Structure-Activity Relationship; Xenopus Proteins

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50 = 0.6 microM) and had the lowest therapeutic index (HC50/IC50 = 0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.

Topics: Amino Acid Sequence; Animals; Antifungal Agents; Antimicrobial Cationic Peptides; Candida albicans; Erythrocytes; Hemolysin Proteins; Hemolysis; Histatins; Humans; Magainins; Molecular Sequence Data; Peptides; Salivary Proteins and Peptides; Xenopus Proteins

In order to obtain peptides having improved antimicrobial activity with low hemolytic effect, a hybrid peptide (CA-MA) composed from cecropin A (1-8) and magainin 2(1-12), and its analogues with amino acid substitutions were designed and synthesized. The antimicrobial activities against bacterial cells and hemolytic activities against human red blood cells were analyzed for each peptide. Secondary structures of the peptides in aqueous solution, 50% trifluoroethanol, and sodium dodecylsulfate micelles were estimated using circular dichroism spectroscopy. The increase in hydrophobicity or alpha-helicity of the peptides correlated with an increase in hemolytic activity rather than antimicrobial activity. The substitution of Leu for Ser at position 16 in CA-MA resulted in a remarkable increase in antimicrobial activity without a significant change in hemolytic activity. Furthermore, the increase in antimicrobial activity of the peptides was not always accompanied by the increase in hemolytic activity.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacillus subtilis; Circular Dichroism; Escherichia coli; Hemolysis; Humans; Magainins; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Xenopus Proteins

Magainin 2 is an antimicrobial peptide found in the skin of Xenopus laevis. To find a reversed peptide comparable to the antibacterial activity of magainin 2 analogs, we have synthesized three reversed analogs, the peptide 53D, 87-ISM and A87-ISM, corresponding to the normal peptide D35, MSI-78 and MSI-78A, respectively. We examined their ability to inhibit the growth of Escherichia coli and Staphylococcus aureus. Among the analogs, the A87-ISM, that is, the reverse of MSI-78A enhanced the amphiphilicity and the alpha-helical tendency of magainin 2, showed not only almost the same antibacterial activity against the bacteria as MSI-78A, but also stronger activity than other magainin 2 analogs. In addition, at the MIC (minimum inhibitory concentration) value, A87-ISM shows no hemolysis to human red blood cells, while both MSI-78 and MSI-78A cause strong hemolysis at the MIC value. This result indicates that a novel reversed peptide comparable or superior to normal magainin 2 analogs is available.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Erythrocytes; Escherichia coli; Hemolysis; Humans; In Vitro Techniques; Magainins; Molecular Sequence Data; Peptides; Staphylococcus aureus; Xenopus Proteins

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Fluoresceins; Gram-Negative Bacteria; Hemolysis; Humans; Lipid Bilayers; Magainins; Microbial Sensitivity Tests; Peptides; Permeability; Phosphatidylcholines; Phosphatidylglycerols; Protein Binding; Protein Structure, Secondary; Structure-Activity Relationship; Xenopus Proteins

Magainin 2 is an antimicrobial peptide isolated from the skin of Xenopus laevis. We have tested the antibacterial activities of normal and reversed magainin 2 analogs against two strains of Helicobacter pylori (ATCC 43526, ATCC 43579), compared with those against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Among these analogs, MSI-78A showed the strongest activity against H. pylori. The MIC (minimum inhibitory concentration) values were almost the same as those against E. coli and S. aureus. No or lesser activity was observed in all the reversed peptides compared to the corresponding normal magainin 2 analogs. Based on the CD (circular dichroism) measurement, the more active peptide tends to show a higher alpha-content. The positively-charged five amino acids (KILKK) positioned at the C terminus on the amphipathical alpha-helical structure play important roles in exerting the strong activity against H. pylori. This indicates that the net charge of the cell surface in H. pylori may be more negative than that of E. coli, though both strains belong to the same genus.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Escherichia coli; Helicobacter pylori; Hemolysis; Humans; Magainins; Molecular Sequence Data; Peptides; Staphylococcus aureus; Structure-Activity Relationship; Xenopus Proteins

Based on the antibacterial activity of 9-phenylnonylamine (pC9a) against Escherichia coli (ATCC29522) and Staphylococcus aureus (ATCC25923), we have further tested the inhibitory ability of the growth of the bacteria by (+/-)1-(4-aminobutyl)-6-benzylindane (PM2) and (+/-)1-benzyl-6-(4-aminobutyl) indane (PM3), that is, two kinds of 1,6-disubstituted indanes. In an in vitro assay, they showed almost the same antibacterial activities against the bacteria as pC9a, as well as that of magainin 2 analogs (i.e., the peptides MSI-78 and 87-ISM), except in the case of 87-ISM against S. aureus. At the MIC (minimum inhibitory concentration) values, however, their killing rate of E. coli is actually quicker than pC9a. This indicates that an indane scaffold, used as a template to mimic a part of the alpha-helical structure of magainin 2, can accelerate the killing rate. At present, however, it is unknown whether either the hydrophobicity or the alpha-helical structure, or both, of the indane scaffold is involved in accelerating the rate. Moreover, these two indanes also showed stronger antibacterial activity against two strains of Helicobacter pylori (ATCC43526, ATCC43579) than either pC9a or magainin 2 related peptides.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Erythrocytes; Escherichia coli; Helicobacter pylori; Hemolysis; Humans; Indans; Magainins; Microbial Sensitivity Tests; Molecular Mimicry; Peptides; Staphylococcus aureus; Xenopus Proteins

A novel antimicrobial peptide, named misgurin, was isolated and characterized from the loach (mudfish), Misgurnus anguillicaudatus. The 21-amino-acid peptide with a molecular mass of 2502 Da was purified to homogeneity using a heparin-affinity column and C18 reverse-phase and gel-permeation high-performance liquid chromatography. The complete amino acid sequence of misgurin, which was determined by an automated amino acid sequencer, was Arg-Gln-Arg-Val-Glu-Glu-Leu-Ser-Lys-Phe-Ser-Lys-Lys-Gly-Ala-Ala-Ala-Arg- Arg-Arg-Lys. Misgurin is a strongly basic peptide which has 5 arginine and 4 lysine residues. Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that misgurin was a novel antimicrobial peptide. Misgurin showed a strong antimicrobial activity in vitro against a broad spectrum of microorganisms without significant hemolytic activity and was about 6 times more potent than magainin 2. Scanning electron microscopy confirmed that the peptide caused damage to the cell membrane by a pore-forming mechanism similar to that of magainin 2. This damage occurred at the minimal inhibition concentration (MIC), but at higher concentration than MIC it lysed the cell.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacillus subtilis; Bacteria; Cell Membrane; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Circular Dichroism; Cypriniformes; Electrophoresis, Polyacrylamide Gel; Hemolysis; Magainins; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Proteins; Sequence Analysis; Xenopus Proteins

The hybrid peptide (CA-ME) derived from cecropin A(1-8) and melittin (1-12) has potent antibacterial and antimalarial activities. Because the N-terminal sequence 1-12 of magainin 2 is similar to melittin(1-12), CA-MA with CA(1-8) and MA(1-12) and their analogues were designed and synthesized. Antitumor activities of these peptides were evaluated using three small cell lung cancer cell lines. Greater antitumor activity was observed when the residues 16, 18 and 19 of the peptide were hydrophobic (Leu or Val), basic (Lys) and basic (Lys), respectively. The IC50 values of the peptides with the residues were 2 to 4 microM. Residue 12 was related to hemolytic activity rather than antitumor activity. Increase in amphipathicity of P4 enhanced hemolytic activity without significant change in antitumor activity. The alpha-helicity of the peptides in a 30 mM sodium dodecyl sulfate solution was more closely correlated to hemolytic activity than antitumor activity.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Antineoplastic Agents; Cell Survival; Circular Dichroism; Hemolysis; Humans; Magainins; Melitten; Molecular Sequence Data; Peptide Fragments; Peptides; Protein Structure, Secondary; Structure-Activity Relationship; Tumor Cells, Cultured; Xenopus Proteins

Magainin peptides, isolated from Xenopus skin, kill bacteria by permeabilizing their cell membranes whereas they do not lyse erythrocytes. To elucidate the rationale for this membrane selectivity, we compared the effects of the membrane lipid composition and the transmembrane potential on the membrane-lytic power of magainin 2 with that of hemolytic melittin. The activity of magainin to zwitterionic phospholipids constituting the erythrocyte surface was extremely weak compared with that of melittin, and acidic phospholipids are necessary for effective action. The presence of sterols reduced the susceptibility of the membrane to magainin. The generation of an inside-negative transmembrane potential enhanced magainin-induced hemolysis. We can conclude that the absence of any acidic phospholipids on the outer monolayer and the abundant presence of cholesterol, combined with the lack of the transmembrane potential, contribute to the protection of erythrocytes from magainin's attack.

Topics: Alamethicin; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Cell Membrane; DNA-Binding Proteins; Erythrocyte Membrane; Gramicidin; Hemolysis; Humans; In Vitro Techniques; Magainins; Melitten; Membrane Lipids; Membrane Potentials; Molecular Sequence Data; Molecular Structure; Peptides, Cyclic; Phospholipids; Sialic Acids; Sterols; Xenopus Proteins

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteriolysis; Cell Membrane Permeability; Cholesterol; Glutamic Acid; Hemolysis; Liposomes; Magainins; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Structure-Activity Relationship; Xenopus laevis; Xenopus Proteins

Novel analogs of the broad-spectrum antimicrobial peptide magainin-2 were obtained by extension of its chain through addition of segments of positively charged amino acids to either its N or its C terminus and by increasing its helicity. The activity of magainin-2 toward American Type Culture Collection strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was most considerably enhanced by these modifications, whereas, in general, its low hemolytic capacity was not or was only slightly affected. The antibacterial potencies of magainin-2 and its derivatives were more evident following decreases of pH from 7.2 to 6 and 5.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Circular Dichroism; Escherichia coli; Hemolysis; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Magainins; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Pseudomonas aeruginosa; Spectrophotometry, Ultraviolet; Staphylococcus aureus; Xenopus Proteins

All-D-magainin-2 was synthesized to corroborate experimentally the notion that the biological function of a surface-active peptide stems primarily from its unique amphiphilic alpha-helical structure. Indeed, the peptide exhibited antibacterial potency nearly identical to that of the all-L-enantiomer. Being highly resistant to proteolysis and non-hemolytic all-D-magainin might have considerable therapeutic importance.

Topics: Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Chymotrypsin; Hemolysis; Humans; Indicators and Reagents; Magainins; Microbial Sensitivity Tests; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Peptides; Protein Conformation; Stereoisomerism; Trypsin; Xenopus Proteins