Magainin Analogues Research Articles

Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

Preparation and evaluation of antibacterial properties of Pexiganan, a Magainin analogue with Broadly-Spectrum Antimicrobial Activity

Introduction: Pexiganan is one of the most popular Maganin-2 analogues, which has broadly- spectrum bactericidal effects against both gram-positive and gram-negative bacteria. In this study, the effects of temperature, pH, and in vitro resistant analysis were evaluated on the antibacterial activity of this peptide. Materials and Methods: Pexiganan recombinant was expressed in P. pastoris GS115, and after its characterization, the cell cytotoxicity characteristics, effects of temperature on antibacterial activity, as well as the emergence of resistance to pexiganan in continuous sub-cultures and MIC of this peptide were investigated against 12 bacterial pathogens. Results: Pexiganan was not cytotoxic to HeLa cell lines. Increasing the temperature had no effect on the zone of inhibition, but increasing the pH caused a significant increase in MIC against S. aureus ATCC 29213 strain. In addition, Pexiganan had inhibitory properties against all the 12 human pathogens (MIC range: 1-32 /g / ml), and no resistance was observed even after 28 sub- cultures. Conclusion: Pexiganan is an AMP with antimicrobial properties against a wide range of susceptible and drug-resistant human pathogens with a very low rate of resistance compared to available antibiotics and is a reliable candidate for further investigation on therapeutic applications. Keywords: Pexiganan; Antimicrobial Peptides; Resistance; Minimum Inhibitory Concentration

Tuning the morphology of mesoscopic structures of porphyrin macrocycles functionalized by an antimicrobial peptide

The aggregation properties of two peptide–porphyrin conjugates were investigated by optical spectroscopy and microscopy imaging with nanometer resolution. Specifically, a tetraphenylporphyrin platform was functionalized by (L)-magainin, a 23-residue long antimicrobial peptide, and by a (L)-magainin analogue differing from the parent peptide by a single residue substitution, [Formula: see text] an Ala vs. Phe replacement in the position 5 of the peptide chain. Spectroscopic and microscopy results show that this single-site substitution has a small effect on the secondary structure attained by the two peptide analogues, but deeply affects the morphology of the mesoscopic structures deposited on hydrophilic mica from methanol/water solutions. In particular, only the Ala-substituted peptide-porphyrin conjugate was shown to be able to form micrometric fibrils, coating homogeneously a hydrophilic mica surface. These results pave the way for potential applications of porphyrin-peptide compounds in localized photodynamic therapy and for designing solid-state stereoselective sensors.

Whole Cell 2H Solid-State NMR of Antimicrobial Peptides Interacting with Cell Envelopes: Role of Lipopolysaccharide

Antimicrobial Peptides (AMPs) have been studied for more than two decades because of their promise to help overcome the problem of resistance to conventional antibiotics. However, AMPs have not been as successful as hoped, likely because we lack a detailed understanding of their mechanisms of action. Many biophysical studies of AMPs are performed in model membrane systems, composed of just one or two lipid components. Such studies have shown that membrane permeabilization is a common mechanism of action. Yet we don’t fully understand their connection between the behavior of AMPs in model lipids, where membrane permeabilization is seen and the behavior in real bacteria, where AMPs are observed to inhibit cell growth. Bacteria have an extra non-lipid component in their cell envelopes. It is thus possible that interactions between AMPs and non-lipid components of the cell envelope are important to their mechanisms of action. Specifically, the focus of this study is to find out if the lipopolysaccharide (LPS) outer membrane layer of Gram-negative bacteria promotes or inhibits AMP-induced membrane disruption. This work employed the magainin analog, MSI-78. We disrupt the lipopolysaccharide layer of Gram-negative bacteria (E.coli) via chelation of the stabilizing divalent cations. Then, we use deuterium NMR of deuterated intact bacteria to observe how AMP-induced acyl chain disruption is affected by LPS layer destabilization. In addition to 2H NMR, we do light microscopy and flow cytometry to confirm the EDTA disruption of LPS.

Magainin-H2 effects on the permeabilization and mechanical properties of giant unilamellar vesicles

Among the potential novel therapeutics to treat bacterial infections, antimicrobial peptides (AMPs) are a very promising substitute due to their broad-spectrum activity and rapid bactericidal action. AMPs strongly interact with the bacterial membrane, and the need to have a correct understanding of the interaction between AMPs and lipid bilayers at a molecular level prompted a wealth of experimental and theoretical studies exploiting a variety of AMPs. Here, we studied the effects of magainin H2 (Mag H2), an analog of the well-known magainin 2 (wt Mag 2) AMP endowed with a higher degree of hydrophobicity, on giant unilamellar vesicles (GUVs) concentrating on its permeabilization activity and the effect on the lipid bilayer mechanical properties. We demonstrated that the increased hydrophobicity of Mag H2 affects its selectivity conferring a strong permeabilization activity also on zwitterionic lipid bilayers. Moreover, when lipid mixtures including PG lipids are considered, PG has a protective effect, at variance from wt Mag 2, suggesting that for Mag H2 the monolayer curvature could prevail over the peptide-membrane electrostatic interaction. We then mechanically characterized GUVs by measuring the effect of Mag H2 on the bending constant of lipid bilayers by flickering spectroscopy and, by using micropipette aspiration technique, we followed the steps leading to vesicle permeabilization. We found that Mag H2, notwithstanding its enhanced hydrophobicity, has a pore formation mechanism compatible with the toroidal pore model similar to that of wt Mag 2.

Effect of Amino Acid Substitutions on Biological Activity of Antimicrobial Peptide: Design, Recombinant Production, and Biological Activity.

Recently, antimicrobial peptides have been introduced as potent antibiotics with a wide range of antimicrobial activities. They have also exhibited other biological activities, including anti-inflammatory, growth stimulating, and anti-cancer activities. In this study, an analog of Magainin II was designed and produced as a recombinant fusion protein. The designed sequence contained 24 amino acid residues (P24), in which Lys, His, Ser residues were substituted with Arg and also, hydrophobic Phe was replaced with Trp. Recombinant production of P24 in Escherichia coli (E. coli) BL21 using pTYB21, containing chitin binding domain and intein sequence at the N-terminus of the peptide gene, resulted in 1 μg mL-1 product from culture. Chitin column chromatography, followed by online peptide cleavage with thiol reducing agent was applied to purify the peptide. Antimicrobial activity was evaluated using five bacteria strains including Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia, E. coli, and Pseudomonas aeruginosa. Designed AMP exhibited promising antimicrobial activities with low minimum inhibitory concentration, in the range of 64-256 µg/mL. P24 showed potent antimicrobial activity preferably against Gram-positive bacteria, and more potent than pexiganan as a successful Magainin II analog for topical infections. In general, further modification can be applied to improve its therapeutic index.

Effects of the peptide Magainin H2 on Supported Lipid Bilayers studied by different biophysical techniques

Given the increasing trend in bacterial antibiotic resistance, research on antimicrobial peptides and their mechanisms of action has become of huge relevance in the last years. Several studies have investigated the effects of a large variety of antimicrobial peptides directly on bacteria or on model lipid bilayers. In the case of model lipid bilayers, different systems are typically exploited; however, different results could be obtained due to the specific properties of the used system. Supported Lipid Bilayers and Giant Unilamellar Vesicles are among the most popular model systems. Here we used Atomic Force Microscopy and fluorescence microscopy to study the interaction of the antimicrobial peptide Magainin H2, an analog of Magainin 2 with increased hydrophobicity, on Supported Lipid Bilayers. We found that, for this kind of model bilayer, due to its strong interaction with the support, the lateral expansion of the membrane induced by the interaction with the peptides is initially inhibited and subsequently proceeds creating new bilayer regions with many defects. This scenario gives rise in Supported Lipid Bilayers to effects like initial increase of lateral pressure, formation of lipid tubes to release this increase, or development of bilayer regions with lower lipid density. Our results highlight that care should be given to the selected model system when studying and comparing the interaction of peptides with other lipid bilayer model systems.

First Example of Kinetic Modelling of Multi-state Membrane Binding, Exapnsion and Disruption for an Antimicrobial Peptide

Antimicrobial peptide disruption of the membrane bilayer involves a series of defined states during binding and insertion, and are all associated with significant changes in the bilayer structure. We recently identified a complex series of bilayer state changes for the binding of an analogue of magainin 2, (Ala-8,13,18)-Magainin 2-amide (MagA), to lipid bilayers. However, the presence of multiple structural states of the membrane prevented the determination of kinetic constants using currently available kinetic models, but this information is necessary to allow the full definition of the mechanism of action. In this study the binding and dissociation of MagA to lipid bilayers with different charge and fluidity properties was studied with dual polarization interferometry, an optical technique capable of measuring real-time simultaneous changes in mass and birefringence (an optical parameter representing bilayer order). The dependence of birefringence vs. mass were categorised into several discrete mechanistic profiles and new multiple-state kinetic models were developed and fitted to the experimental data, with a three-state model with lateral bilayer expansion providing distinctly superior fits to simpler model types. Overall this is the first study to quantitatively analyse complex peptide-membrane binding data in terms of specific states of membrane disruption by an antimicrobial peptide.

Bactericidal Peptidomimetic Polyurethanes with Remarkable Selectivity against Escherichia coli.

The increasing incidence of drug-resistant strains of bacteria necessitates the development of new classes of antimicrobials. Host defense peptides, also known as antimicrobial peptides, are promising in this regard but have several drawbacks. Herein, we show that peptidomimetic polyurethanes with pendant functional groups that mimic lysine and valine amino acid residues have high antibacterial activity against Gram negative Escherichia coli, yet are less effective against Gram positive Staphylococcus aureus. All the polyurethanes designed in this study display high bactericidal activity against E. coli, whereas the polyurethanes with high concentrations of lysine mimicking functional groups display minimal cytotoxicity toward mammalian cells. Control experiments with pexiganan, an analogue of the host defense peptide magainin, showed that the polyurethanes described here have high bactericidal activity, while having comparable hemocompatibility and lower mammalian cell toxicity. Overall, the results point to an encouraging new class of peptidomimetic synthetic polymers with selective bactericidal activity to E. coli and low mammalian cell toxicity.

Utilisation of peptides against microbial infections - a review.

The emergence of resistance in microorganisms on a global scale has made it necessary to search for new antimicrobial factors. Antimicrobial peptides (AMPs) seem to meet these expectations. AMPs are produced by bacteria, viruses, plants, and animals, and may be considered as a new class of drugs intended for the prophylaxis and treatment of both systemic and topical infections. The aim of this study is to review the results of studies on the use of peptides to combat infections in vivo. Antimicrobial peptides may be applied topically and systemically. Among the peptides used topically, a very important area for their application is ophthalmology. AMPs in ophthalmology may be used mainly for the protection of contact lenses from ocular pathogens. Many AMPs are in clinical trials for application in the therapy of local infections. There may be mentioned such preparations as: pexiganan (magainin analogue), MX-226 (based on indolicidin), NEUPREX (isolated from human BPI (bactericidal/permeability-increasing) protein), IB-367 (variant of porcine protegrin), P113 (based on histatin), daptomycin, polymyxins, as well as peptidomimetics. In the combat against systemic infections are used such peptides as: P113D (modified P113 peptide containing D-amino acids), colistin, peptoids, and peptides containing non-typical amino acids or non-peptide elements. AMPs are also used as antiprotozoal, antifungal, antitoxic and immunostimulatory agents. The limitations in the use of peptides in the treatment of infections, such as susceptibility to proteolysis, and resistance of microorganisms to the peptides, are also discussed. AMPs are a promising strategy in the fight against microbial infections.

In Vitro Activity of Pexiganan and 10 Comparator Antimicrobials against 234 Isolates, Including 93 Pasteurella Species and 50 Anaerobic Bacterial Isolates Recovered from Animal Bite Wounds.

Animal bite wounds affect more than 5 million Americans annually, resulting in 300,000 emergency department visits, 10,000 hospitalizations, and an untold number of physician office visits. Various forms of topical therapy are empirically self-employed by many patients prior to seeking medical attention. Pexiganan, a 22-amino-acid synthetic cationic analogue of the peptide magainin II, acts by selectively damaging bacterial cell membranes. We determined the MICs for pexiganan and other antimicrobial agents often used for treatment of bite wounds. Most isolates were from U.S. patients, and ∼10% were from European and Canadian patients. The comparator antimicrobials studied were penicillin, amoxicillin-clavulanate, piperacillin-tazobactam, meropenem, clindamycin, doxycycline, moxifloxacin, ceftriaxone, linezolid, and metronidazole. The MIC90s of pexiganan were 32 μg/ml (against Pasteurella multocida subsp. multocida), 16 μg/ml (P. multocida subsp. septica, Pasteurella canis, and Pasteurella dagmatis), 8 μg/ml (Pasteurella stomatis), 8 μg/ml (Eikenella corrodens), 2 μg/ml (Neisseria weaveri, Neisseria zoodegmatis, and Moraxella canis-Moraxella lacunata group), 16 μg/ml (Bergeyella zoohelcum), 64 μg/ml (Bacteroides pyogenes), 4 μg/ml (Fusobacterium russii), 32 μg/ml (Fusobacterium canifelinum), and 64 μg/ml (Prevotella heparinolytica). The concentration of pexiganan in the cream used was 8,000 μg/ml, more than 60 to 100 times the highest MIC obtained. Pexiganan exhibited a broad range of antimicrobial activity, showing potential for treating animal bite infections. A clinical trial seems warranted.

The synthetic antimicrobial peptide pexiganan and its nanoparticles (PNPs) exhibit the anti-helicobacter pylori activity in vitro and in vivo.

The aim of this study was to probe the potential anti-H. pylori activity of the synthetic antimicrobial peptide pexiganan, which is an analog of the peptide magainin, and its nanoparticles (PNPs) that were prepared in our laboratory. To compare their antibacterial effects in vitro and in vivo, studies of H. pylori growth inhibition, kinetics and resistance assays were undertaken. The gastric mucoadhesive efficiency and H. pylori clearance efficiency of pexiganan and PNPs were evaluated in rats and mice infected with H. pylori. The eradication of H. pylori was determined using urease tests and a microbial culture method. We observed that PNPs adhered to gastric mucosa more effectively owing to a prolonged stay in the stomach, which resulted in a more effective H. pylori clearance. In addition, PNPs had greater anti-H. pylori effect than pexiganan in infected mice. The amount of pexiganan required to eradicate H. pylori was significantly less using PNPs than the corresponding pexiganan suspension. The results confirmed that PNPs improved peptide stability in the stomach and more effectively eradicated H. pylori from mice stomachs than pexiganan.

Design of protease-resistant pexiganan enhances antileishmanial activity

Antimicrobial peptides are increasingly being explored as alternative agents for therapy against the parasitic protozoan Leishmania. Previously, we reported that the synthetic magainin analog, pexiganan, induced apoptosis of surface protease-deficient Leishmania. Here, we report the development of an arginine-rich variant of this peptide which has reduced protease susceptibility and enhanced activity against wild type Leishmania in vitro. This peptide induces calcium delocalization and caspase 3/7 activity indicative of apoptosis, demonstrating that structural modification of pexiganan leads to drastic changes in biologic activity against Leishmania.

Charge distribution and imperfect amphipathicity affect pore formation by antimicrobial peptides

Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analog MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136ns to 216ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melittin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable.

The Antimicrobial Peptide Analog Magainin-H2 Induces Changes in the Phase Behavior and Lateral Organization of Supported Bilayers During Liquid-Crystalline/Gel Phase Coexistence

Antimicrobial peptides (AMPs) disrupts membrane function in bacterial cells. The mechanism for this is still the subject of diverse studies. It is generally believed that AMPs enriched in cationic residues are attracted to the anionic bacterial membrane through electrostatic interactions, but the mode of membrane disruption appears to vary among AMPs. For some types of AMPs, for example Magainin, protegrins, alamethicin and melittin, the adhesion to the bilayer generates a series of configurational changes leading to pore formation. After binding, these peptides accumulate on the cell surface, adopting an alpha helical configuration. This accumulation leads to membrane thinning, followed by the translocation of the peptide across the membrane and the formation of a pore induced by aggregation. We are interested in exploring how the physical state of the membrane and its lateral organization is influenced by AMP binding. In order to accomplish this we visualize DMPC supported bilayers close to a liquid-crystalline/gel phase transition temperature by atomic force microscopy in real time as Magainin-H2 (Mag-H2), a magainin analog that binds to zwitterionic membranes, is added. Close to the phase transition temperature of 24°C coexisting liquid-crystalline/gel phase domains are observed with a height difference of approximately 1.5 nm. After addition of the peptide we observe morphological changes in the domain structure and an increase in the height difference between the two phases, eventually leading to amorphous hole formation. Laurdan generalized polarization measurements indicate that the presence of Mag-H2 increases the phase transition temperature of DMPC, indicating an increase in membrane packing close to the phase transition temperature. The results are relevant in pointing to a mechanism for membrane perturbation by Mag’H2 through changes in the lateral organization of the membrane.

In-vitro effects of the antimicrobial peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells

BackgroundResearch on antimicrobial cationic peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an antimicrobial cationic peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of cationic antimicrobial peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard.MethodsImmunocytochemical (ICC) analyses using confocal microscopy (n = 6 for each treatment sub-group) and Western blot (WB) method (n = 5 for each treatment sub-group) of CTB differentiation based on synthesis of beta-hCG and hPL, and apoptosis based on apoptosis-associated cytokeratin 18 neo-epitope (CK18f) were performed for CTB isolated from human first trimester placental villi and grown in serum-free primary culture for 24 h, 48 h and 96 h on rat-tail collagen with and without AMA (1000 ng/ml). Moreover, secretion of beta-hCG and hPL into conditioned media from isolated CTB grown in vitro for 24 h, 48 h and 96 h (n = 6/each sub-group) with and without AMA was examined using enzyme immunoassays. Furthermore, TUNEL assay, and cell viability based on LDH leakage into medium (n = 6/each sub-group) were assessed to examine the phenomenon of cell death with time and administration of AMA.ResultsCTB in serum-free primary culture showed increased (P < 0.05) level of synthesis and secretion of beta-hCG and hPL with time, and higher (P < 0.05) level of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h of culture. Exposure of CTB to AMA resulted in lower (P < 0.05) level of synthesis and secretion of beta-hCG and hPL, as well as, an increase (P < 0.05) of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h as compared to the control treatment.ConclusionsAdministration of AMA resulted in attenuation of differentiation, enhancement in apoptosis and loss of viability in early placental villi trophoblast cells in primary culture. Thus, it appears that administration of alpha-helical AMP may adversely affect the process of placentation and pregnancy outcome.

Amphipathic Antimicrobial Peptides – from Biophysics to Therapeutics?

Amphipathic peptides are accommodated within the diffuse gradient of polarity that characterizes the interfacial regions of phospholipid bilayer membranes. Interfacial membrane interactions are key to the diverse biological functions and activities of these peptides, which encompass a large class of antimicrobial peptides, including the helical peptides magainin, melittin, and RTA3 derived from the commensal bacterium Streptococcus mitis. For these peptides in vitro efficacy (high antimicrobial activity with minimal mammalian cell toxicity, equivalent to high potential therapeutic index; PTI), can be broadly understood in relation to the thermodynamics of interfacial binding and membrane disruption in membranes having surface charges that correspond to bacterial and mammalian cell membranes, respectively. Peptides with disrupted amphipathicity resulting from a positively charged amino acid residue on the non-polar helix face, can have greatly enhanced PTI, although a balance of amphipathicity, hydrophobicity and positive charge is required for retention of high antimicrobial activity. These observations are illustrated with recent examples from the literature, and studies on RTA3 and magainin analogues from our laboratories. Despite the identification and optimisation of peptides with very good PTI, a focus on addressing toxicity upon systemic administration and poor in vivo efficacy is likely to be required to translate growing understanding of the relationships between peptide interfacial activity and effects on cells, into novel systemic therapeutics.

Antimicrobial peptides bind more strongly to membrane pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.

Agrobacterium-Mediated Transformation of Pineapple (Ananas comosus L. Merr.) Leaf Bases with MSI-99, a Magainin Analogue

Leaf bases excised from in vitro derived shoots of pineapple (Ananas comosus LMerr. cv. Queen) were transformed with Agrobacterium tumefaciens strain EHA 105 harboring the pMSI168 plasmid with MSI-99, a magainin analogue, by co-cultivating for three days in the dark on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962). The leaf bases produced callus and/or multiple shoots upon transfer to MS medium supplemented with NAA (1.8 mg/l), IBA (2.0 mg/l), kinetin (2.0 mg/l), cefotaxime (400 mg/l) and kanamycin (50 mg/l) after six to eight weeks. Six percent of the leaf bases produced friable callus, and only two percent produced multiple shoots directly without intervening callus formation. Putatively transformed shoots (1–2 cm) were selected and multiplied on medium of the same composition, and elongated shoots (5 cm) were rooted on liquid White's (1954) medium supplemented with NAA (0.1 mg/l ), IBA (0.4 mg/l), cefotaxime (400 mg/l), and kanamycin (100 mg/l). A few of the transgenic plants were grown to maturity in the greenhouse. The rooted plants were analyzed through PCR, Southern analysis of the PCR products, and reverse transcription (RT)-PCR. The results clearly confirmed the integration and expression of MSI-99. All the plants were morphologically normal and set fruits. To the best of our knowledge this is the first report on the transformation of pineapple with MSI-99, a magainin analogue.

Magainin 2 in Action: Distinct Modes of Membrane Permeabilization in Living Bacterial and Mammalian Cells

Interactions of cationic antimicrobial peptides with living bacterial and mammalian cells are little understood, although model membranes have been used extensively to elucidate how peptides permeabilize membranes. In this study, the interaction of F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS), an equipotent analogue of magainin 2 isolated from the African clawed frog Xenopus laevis, with unfixed Bacillus megaterium and Chinese hamster ovary (CHO)-K1 cells was investigated, using confocal laser scanning microscopy. A small amount of tetramethylrhodamine-labeled F5W-magainin 2 was incorporated into the unlabeled peptide for imaging. The influx of fluorescent markers of various sizes into the cytosol revealed that magainin 2 permeabilized bacterial and mammalian membranes in significantly different ways. The peptide formed pores with a diameter of ∼2.8nm (< 6.6nm) in B. megaterium, and translocated into the cytosol. In contrast, the peptide significantly perturbed the membrane of CHO-K1 cells, permitting the entry of a large molecule (diameter, >23nm) into the cytosol, accompanied by membrane budding and lipid flip-flop, mainly accumulating in mitochondria and nuclei. Adenosine triphosphate and negatively charged glycosaminoglycans were little involved in the magainin-induced permeabilization of membranes in CHO-K1 cells. Furthermore, the susceptibility of CHO-K1 cells to magainin was found to be similar to that of erythrocytes. Thus, the distinct membrane-permeabilizing processes of magainin 2 in bacterial and mammalian cells were, to the best of our knowledge, visualized and characterized in detail for the first time.