Magainin Research Articles

Hybrid bio-nanoporous peptide loaded-polymer platforms with anticancer and antibacterial activities.

In this study, hybrid bio-nanoporous peptides loaded onto poly(N-isopropylacrylamide-co-butylacrylate) (pNIPAM-co-BA) coatings were designed and obtained via matrix-assisted pulsed laser evaporation (MAPLE) technique. The incorporation of cationic peptides magainin (MG) and melittin (Mel) and their combination was tailored to target synergistic anticancer and antibacterial activities with low toxicity on normal mammalian cells. Atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy as well as contact angle and surface energy measurements revealed the successful and functional incorporation of both the peptides within porous polymeric nanolayers as well as surface modifications (i.e. variation in the pore size diameter, surface roughness, and wettability) after Mel, MG or Mel-MG incorporation compared to pNIPAM-co-BA. In vitro testing revealed the impairment of biofilm formation on all the hybrid coatings while testing with S. aureus, E. coli and P. aeruginosa. Moreover, MG was shown to modulate the effect of Mel in the combined Mel-MG extract formulation released via pNIPAM-platforms, thus significantly reducing cancer cell proliferation through apoptosis/necrosis as revealed by flow cytometry analysis performed in vitro on HEK293T, A375, B16F1 and B16F10 cells. To the best of our knowledge, Mel-MG combination entrapped in the pNIPAM-co-BA copolymer has not yet been reported as a new promising candidate with anticancer and antibacterial properties for improved utility in the biomedical field. Mel-MG incorporation compared to pNIPAM-co-BA in in vitro testing revealed the impairment of biofilm formation in all the hybrid formulations.

Exploring and characterizing the sensitivity of Bdellovibrio bacteriovorus against different stressors.

Given the increasing concern of antibiotic resistance, scientists have been pushed to look for new and interesting ways to kill bacteria. One area of research focuses on using bacterial predators, such as the Gram-negative predatory bacteria Bdellovibrio bacteriovorus, which has been proposed as a “living antibiotic.” B. bacteriovorus consumes other Gram-negative bacteria, burrowing into the periplasmic space and digesting the prey from the inside before releasing several progeny cells. Using a host-independent mutant, we have exposed B. bacteriovorus to various physiologically relevant stressors, such as temperature and the presence of a model antimicrobial peptide (AMP), used as part of the innate immune response. We first noticed that B. bacteriovorus cultures grown at 37°C become dark yellow after one day of growth, while B. bacteriovorus grown at 30°C remain light yellow even after several days of growth. The dark yellow color is caused by the production of ubiquinone-8 (UQ8), an essential member of the bacterial electron transport chain (ETC).We also found that B. bacteriovorus grown at 37°C has increased sensitivity to the AMP magainin 2 (MAG2). MAG2 kills bacteria by forming pores in bacterial membranes, which will disrupt the bacterial ETC, and lead to the production of reactive oxygen species. We monitored the autofluorescence of B. bacteriovorus during MAG2 treatment and observed an oxidative burst directly after adding MAG2.With the increased production of UQ8, B. bacteriovorus grown at 37°C could have a difference in membrane fluidity, making it easier for MAG2 to insert into the cell membrane, disrupting the ETC and killing the cell. By determining how we can manipulate its stress response, we can engineer B. bacteriovorus to become a true “living antibiotic.”

Characterizing the role of the cell wall in bacterial death caused by treatment with antimicrobial peptides.

The rise in pathogenic bacteria becoming resistant to all commercially available antibiotics has compelled scientists to develop new antimicrobial compounds while also identifying effective bacterial targets. Antimicrobial peptides (AMPs), which are part of many different species’ nonspecific immune responses, have shown promising results in eliminating invading bacteria. Several classes of AMPs selectively target bacterial membranes, which have been identified prior as a good target for new antibiotics. The cationic AMP magainin 2 (MAG2) folds into an alpha helix upon contact with the negatively charged bacterial cell wall. Several folded subunits come together to form pores in the bacterial membranes, which leads to the uncontrolled release of cytosol and, eventually, cell death. Using minimum inhibitory concentration (MIC) assays, we found that Staphylococcus epidermidis (Gram positive) was more susceptible to MAG2 than E. coli (Gram negative). We used propidium iodide uptake assays and autofluorescence to evaluate the role of the cell wall in MAG2 activity and bacterial death. We found that despite the increased sensitivity of E. coli to MAG2, pore formation takes longer to reach equilibrium in E. coli than in the S. epidermidis, which is likely due to the difference in the cell walls. We also used fluorescence microscopy and growth assays in order to determine when cell death truly occurred. We found that S. epidermidis was able to regrow at sub-MIC concentrations which shows us that cell membrane integrity is not an adequate way to define cell death.

Formation of β-Strand Oligomers of Antimicrobial Peptide Magainin 2 Contributes to Disruption of Phospholipid Membrane.

The antimicrobial peptide magainin 2 (M2) interacts with and induces structural damage in bacterial cell membranes. Although extensive biophysical studies have revealed the interaction mechanism between M2 and membranes, the mechanism of membrane-mediated oligomerization of M2 is controversial. Here, we measured the synchrotron-radiation circular dichroism and linear dichroism (LD) spectra of M2 in dipalmitoyl-phosphatidylglycerol lipid membranes in lipid-to-peptide (L/P) molar ratios from 0–26 to characterize the conformation and orientation of M2 on the membrane. The results showed that M2 changed from random coil to α-helix structures via an intermediate state with increasing L/P ratio. Singular value decomposition analysis supported the presence of the intermediate state, and global fitting analysis revealed that M2 monomers with an α-helix structure assembled and transformed into M2 oligomers with a β-strand-rich structure in the intermediate state. In addition, LD spectra showed the presence of β-strand structures in the intermediate state, disclosing their orientations on the membrane surface. Furthermore, fluorescence spectroscopy showed that the formation of β-strand oligomers destabilized the membrane structure and induced the leakage of calcein molecules entrapped in the membrane. These results suggest that the formation of β-strand oligomers of M2 plays a crucial role in the disruption of the cell membrane.

Extracellular Polymeric Substance Protects Some Cells in an Escherichia coli Biofilm from the Biomechanical Consequences of Treatment with Magainin 2.

Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS.

Cell-penetrating peptides in oncologic pharmacotherapy: A review

Cancer is the second leading cause of death in the world and its treatment is extremely challenging, mainly due to its complexity. Cell-Penetrating Peptides (CPPs) are peptides that can transport into the cell a wide variety of biologically active conjugates (or cargoes), and are, therefore, promising in the treatment and in the diagnosis of several types of cancer. Some notable examples are TAT and Penetratin, capable of penetrating the central nervous system (CNS) and, therefore, acting in cancers of this system, such as Glioblastoma Multiforme (GBM). These above-mentioned peptides, conjugated with traditional chemotherapeutic such as Doxorubicin (DOX) and Paclitaxel (PTX), have also been shown to induce apoptosis of breast and liver cancer cells, as well as in lung cancer cells, respectively. In other cancers, such as esophageal cancer, the attachment of Magainin 2 (MG2) to Bombesin (MG2B), another CPP, led to pronounced anticancer effects. Other examples are CopA3, that selectively decreased the viability of gastric cancer cells, and the CPP p28. Furthermore, in preclinical tests, the anti-tumor efficacy of this peptide was evaluated on human breast cancer, prostate cancer, ovarian cancer, and melanoma cells in vitro, leading to high expression of p53 and promoting cell cycle arrest.Despite the numerous in vitro and in vivo studies with promising results, and the increasing number of clinical trials using CPPs, few treatments reach the expected clinical efficacy. Usually, their clinical application is limited by its poor aqueous solubility, immunogenicity issues and dose-limiting toxicity. This review describes the most recent advances and innovations in the use of CPPs in several types of cancer, highlighting their crucial importance for various purposes, from therapeutic to diagnosis. Further clinical trials with these peptides are warranted to examine its effects on various types of cancer.

Individual Roles of Peptides PGLa and Magainin 2 in Synergistic Membrane Poration.

Synergy between antimicrobial peptides PGLa and Magainin 2 (MAG2) provides an efficient way to enhance their antimicrobial ability. However, the underlying molecular mechanism of such synergy, especially the individual roles of each peptide, remains poorly understood. We combined a giant unilamellar vesicle leakage assay, in situ interfacial photovoltage testing, and molecular dynamics to investigate membrane poration under the action of PGLa, MAG2, or a PGLa/MAG2 mixture. Our results clearly show the different membrane action modes of the three systems and demonstrate the importance of forming PGLa-MAG2 heterodimers in the membrane poration process. PGLa inserted into and extracted from a membrane rapidly and continually with minimal aggregation and produced only transient, small pores. In contrast, MAG2 peptides tended to aggregate together on the membrane surface or only shallowly embed in the membrane. Additionally, the PGLa and MAG2 residues were well integrated into the membrane via the formation of PGLa-MAG2 heterodimers. The membrane defect produced by the rapid insertion of PGLa was stabilized by MAG2, which further recruited other peptides for the formation of PGLa-MAG2 heterodimers and even heterodimer clusters. Growth in pore size then occurred in a step-by-step process involving the formation and assembly of heterodimer clusters within the membrane. Our results provide insight into the complicated synergy that occurs between PGLa and MAG2 during membrane poration and will assist in the design of new antimicrobial peptides.

Qualitative and Quantitative Changes to Escherichia coli during Treatment with Magainin 2 Observed in Native Conditions by Atomic Force Microscopy.

The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

The "pre-assembled state" of magainin 2 lysine-linked dimer determines its enhanced antimicrobial activity.

Antimicrobial peptides (AMPs) are alternatives to conventional antibiotics against multi-drug resistant bacteria with low potential for developing microbial resistance. The design of such molecules requires understanding of the mechanisms of action, particularly the interaction with bacteria cell membranes. In this work, we determine the mechanism responsible for the higher activity against Escherichia coli of the C-terminal lysine dimer of magainin 2, (MG2)2K, in comparison to the monomeric peptide magainin 2 (MG2). Langmuir monolayers and vesicles made with the E. coli lipid extract were used to address the two possible states for the peptide-membrane interaction, namely the "binding state" and "pore state", respectively. The incorporation of MG2 and (MG2)2K in lipid monolayers at the air-water interface caused slight differences in surface pressure isotherms and polarization-modulated infrared reflection absorption (PM-IRRAS) spectra, and therefore the difference in activity is not associated with the binding state. In contrast, large differences were observed in the leakage experiments where (MG2)2K was shown to disrupt the large unilamellar vesicles to a much higher extent owing to efficient pore formation. The binding and penetration of MG2 and (MG2)2K were also probed with molecular dynamics (MD) simulations for bilayers made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE:POPG). (MG2)2K forms disordered toroidal pores at a significant lower concentration than for MG2. In summary, the combination of experimental and computational simulation results indicated that the "pre-assembling state" of (MG2)2K dimer leads to a reduced number of molecules and shorter time being required to kill E. coli.

Computational study of solution behavior of magainin 2 monomers

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity – the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.

Molecular Mechanism of Synergy between the Antimicrobial Peptides PGLa and Magainin 2 in Membranes

The two antimicrobial peptides PGLa and magainin 2 (MAG2), both found in the skin of the African frog Xenopus laevis, are known to exhibit strong synergistic effects in bacterial killing, lipid vesicle leakage, and other membrane-related activities [1]. Here, we studied the synergy of the two peptides in an attempt to find the molecular mechanism behind their synergistic effect [2]. A large number of mutants of the two peptides were investigated, and the synergy tested was using three complementary methods: (i) a checkerboard assay against two bacteria; (ii) a vesicle leakage assay; and (iii) 15N-NMR, showing the insertion of PGLa into a transmembrane orientation (suggesting the formation of a pore) in the presence of MAG2. Several mutations were identified that reduced or inhibited the synergy of the peptides, and a single mutation on PGLa from Gly to Ala was enough to completely abolish synergy. At the same time, the designer-made and related but completely non-synergistic peptide MSI-103 could be modified by two point mutations to become strongly synergistic with MAG2. Also in this case Gly residues were involved, indicating that a GxxxG motif on PGLa is critical and necessary for synergy. Based on these results, a 3D model could be developed of the PGLa-MAG2 complex in the membrane that is synergistically active. An oligomeric pore is likely formed from antiparallel dimers of transmembrane PGLa peptides, wherein each PGLa molecule makes contact to a MAG2 peptide that is aligned on the membrane surface.

Molecular mechanism of synergy between the antimicrobial peptides PGLa and magainin 2

PGLa and magainin 2 (MAG2) are amphiphilic α-helical membranolytic peptides from frog skin with known synergistic antimicrobial activity. By systematically mutating residues in the two peptides it was possible to identify the ones crucial for the synergy, as monitored by biological assays, fluorescence vesicle leakage, and solid-state 15N-NMR. Electrostatic interactions between anionic groups in MAG2 and cationic residues in PGLa enhance synergy but are not necessary for the synergistic effect. Instead, two Gly residues (7 and 11) in a so-called GxxxG motif in PGLa are necessary for synergy. Replacing either of them with Ala or another hydrophobic residue completely abolishes synergy according to all three methods used. The designer-made peptide MSI-103, which has a similar sequence as PGLa, shows no synergy with MAG2, but by introducing two Gly mutations it was possible to make it synergistic. A molecular model is proposed for the functionally active PGLa-MAG2 complex, consisting of a membrane-spanning antiparallel PGLa dimer that is stabilized by intimate Gly-Gly contacts, and where each PGLa monomer is in contact with one MAG2 molecule at its C-terminus.

Effects of Lipid Composition, Peptide Charge, and Molecularity on the Structure of Antimicrobial Peptide Transmembrane Pores

The structure of transmembrane pores formed by antimicrobial and cell-penetrating peptides depends on the properties of the membrane and the peptide. To examine the effect of lipid composition, we simulated magainin 2 (MAG2) antiparallel tetramers in DMPC, DMPG and DMPC/PG 1:1 bilayers. Together with the results from our previous work (1), we observe that the MAG2 pore size initially decreases as the anionic content of the membrane increases. However, in pure DMPG the observed pore is similar in size to that in DMPC, but with considerably more unfolded peptides. We also simulated a PGLa parallel tetramer in POPE/PG 3:1 and observed that 1-2 monomers either adopt or tend to adopt a surface orientation during the 5 μs of the simulation, in partial agreement with NMR data (2).We explored the effect of molecularity by simulations of hexamers and octamers of melittin and octamers of MAG2 and comparison with previous tetramer results (1,3). The results show a stable wide pore for the MAG2 octamer, with all the monomers involved in the formation and support of the water channel. On the contrary, in both melittin oligomers the number of peptides in contact with the pore is reduced during the simulation. Finally, we explored the effect of peptide charge by comparing simulations of hexamers of melittin (charge +5) and its gain-of-function variant MELP5 (charge +2) (4). Whereas the melittin hexamer broke up into two trimers, one surrounding a pore and one dry, the MELP5 hexamer simulation resulted in a stable wide pore.(1) Pino-Angeles A, Leveritt JM 3rd and Lazaridis T. PloS Comput Biol (2016) 12(1):e1004570(2) Stranberg E, Zerweck J et al. Biophys J (2013) 104(6):L9-11(3) Leveritt JM 3rd, Pino-Angeles A and Lazaridis T. Biophys J (2015) 108(10):2424-6(4) Wiedman G, Fuselier T et al. J Am Chem Soc (2014) 136(12):4724-31

2H-NMR and MD Simulations Reveal Membrane-Bound Conformation of Magainin 2 and Its Synergy with PGLa

Magainin 2 (MAG2) and PGLa are two α-helical antimicrobial peptides found in the skin of the African frog Xenopus laevis. They act by permeabilizing bacterial membranes and exhibit an exemplary synergism. Here, we determined the detailed molecular alignment and dynamical behavior of MAG2 in oriented lipid bilayers by using 2H-NMR on Ala-d3-labeled peptides, which yielded orientation-dependent quadrupolar splittings of the labels. The amphiphilic MAG2 helix was found to lie flat on the membrane surface in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), as expected, with a tilt angle close to 90°. This orientation fits well with all-atom molecular-dynamics simulations of MAG2 performed in DMPC and DMPC/DMPG. In the presence of an equimolar amount of PGLa, the NMR analysis showed that MAG2 becames tilted at an angle of 120°, and its azimuthal rotation angle also changes. Since this interaction was found to occur in a concentration range where the peptides per se do not interact with their own type, we propose that MAG2 forms a stable heterodimer with PGLa. Given that the PGLa molecules in the complex are known to be flipped into a fully upright orientation, with a helix tilt close to 180°, they must make up the actual transmembrane pore. We thus suggest that the two negative charges on the C-terminus of the obliquely tilted MAG2 peptides neutralize some of the cationic groups on the upright PGLa helices. This would stabilize the assembly of PGLa into a toroidal pore with an overall reduced charge density, which could explain the mechanism of synergy.

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

Homo- and heteromeric interaction strengths of the synergistic antimicrobial peptides PGLa and magainin 2 in membranes.

PGLa and magainin 2 (MAG2) are amphiphilic α-helical frog peptides with synergistic antimicrobial activity. In vesicle leakage assays we observed the strongest synergy for equimolar mixtures of PGLa and MAG2. This result was consistent with solid-state (15)N-NMR data on the helix alignment in model membranes. The Hill coefficients determined from the vesicle leakage data showed that the heterodimeric (PGLa-MAG2) interactions were stronger than the homodimeric (PGLa-PGLa and MAG2-MAG2) interactions. This result was also reflected in the free energy of dimerization determined from oriented circular dichroism and quantitative solid-state (19)F-NMR analysis.

Influence of hydrophobic residues on the activity of the antimicrobial peptide magainin 2 and its synergy with PGLa.

Magainin 2 (MAG2) and PGLa are two related antimicrobial peptides found in the skin of the African frog Xenopus laevis with a pronounced synergistic activity, which act by permeabilizing bacterial membranes. To probe the influence of hydrophobic peptide-lipid and peptide-peptide interactions on the antimicrobial activity and on synergy, the sequence of MAG2 was modified by replacing single amino acids either with a small alanine or with the stiff and bulky hydrophobic 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine side chain. The minimum inhibitory concentration of 14 MAG2 analogs was strongly influenced by these single substitutions: the antimicrobial activity was consistently improved when the hydrophobicity was increased on the hydrophobic face of the amphiphilic helix, while the activity decreased when the hydrophobicity was reduced. The synergy with PGLa, on the other hand, was rather insensitive to mutations of hydrophobic residues. It thus seems that the antimicrobial effect of MAG2 on its own depends strongly on the hydrophobicity of the peptide, while the synergy with PGLa does not depend on the overall hydrophobicity of MAG2.

Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli

The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 104CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6CFU per 25g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in Membranes Depends on the Lipid Spontaneous Curvature

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state 15N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.

Modulation of Elastic Properties through Unsaturated Lipid Content as a Mechanism for Inducing Resistance to Amphiphilic Antimicrobial Peptides

We explore whether changes in fatty acid composition in bacterial membrane models lead to inhibition of amphiphilic antimicrobial peptide-(AMPs) through the modulation of the membrane elastic properties. Specifically, we examined whether changes in the unsaturated/saturated fatty acid ratio, which is controlled by bacteria, inhibit AMP function. In phosphatidylglycerol (PG) lipid bilayers we observe a 10-fold increase in resistance to Magainin-2 (MAG) in POPG (16:0/18:1) vs. DPPG (16:0/16:0) bilayers and a 20-fold increase in resistance in DOPG (18:1/18:1) vs. DPPG bilayers, as measured by calcein release at 45 C (where lipids are in the liquid-crystalline phase). By analyzing the leakage kinetics, we find that the activation energy for pore formation in DPPG/POPG mixtures increases linearly with POPG content, confirming that unsaturation is energetically unfavorable for pore formation. Laurdan polarization (GP) measurements correlate well with MAG resistance, suggesting that head group spacing is an indicator of resistance (increased head-group spacing could allow for more peptides to partition into the head group region, instead of becoming inserted into the bilayer as bilayer-spanning monomers). The changes in potency cannot be explained solely by the increased head group spacing, however. We therefore performed leakage experiments using known modifiers of membrane elastic properties (Triton-X, capsaicin) that both reduce bilayer stiffness but cause opposite changes in curvature. Bilayer softening plays a role in decreasing MAG's potency, whereas the curvature alters only the leakage kinetics, not potency. Based on previous studies, we propose that membrane softening somehow makes it more difficult for MAG to reach a critical concentration necessary to produce pore formation⎯maybe because the energetics of lateral association among bilayer-spanning MAG monomers favors the monomers in softer bilayers. Unsaturated lipids may therefore influence MAG potency by modulating membrane stiffness.