A new group of antifungal and antibacterial lipopeptides derived from non-membrane active peptides conjugated to palmitic acid

We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K(4)X(7)W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections.     

Conjugation of a magainin analogue with lipophilic acids controls hydrophobicity, solution assembly, and cell selectivity

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.     

Preassembly of membrane-active peptides is an important factor in their selectivity toward target cells

Membrane-active peptides comprise a large group of toxins used in the defense and offense systems of all organisms including plants and humans. They act on diverse targets including microorganisms and mammalian cells, but the factors that determine their target cell selectivity are not yet clear. Here, we tested the role of peptide length and preassembly on the ability of diastereomeric cationic antimicrobial peptides to discriminate among bacteria, erythrocytes, and fungal cells, by using peptides with variable lengths (13, 16, and 19 amino acids long) and their covalently linked pentameric bundles. All the bundles expressed similar potent antifungal activity (minimal inhibitory concentration of 0.2-0.3 microM) and high antimicrobial activity. Hemolytic activity was also observed at concentrations higher than those required for antifungal activity. In contrast, all the monomers showed length-dependent antimicrobial activity, were less active toward bacteria and fungi, and were devoid of hemolytic activity. BIAcore biosensor experiments revealed a approximately 300-fold increase in peptide-membrane binding affinity between the 13- and 19-residue monomers toward zwitterionic (egg phosphatidylcholine (PC)/egg spingomyelin (SM)/cholesterol) vesicles. All the monomeric peptides display a similar high affinity to negatively charged (E. coli phosphatidylethanolamine (PE)/egg phosphatidylglycerol (PG)) vesicles regardless of their length. In contrast, irrespective of the size of the monomeric subunit, all the bundles bind irreversibly and strongly disrupt both PC/SM/cholesterol and PE/PG membranes. Attenuated total reflectance Fourier-transform infrared spectroscopy revealed that peptide assembly also affects structure as observed by an increased alpha-helical and beta-sheet content in membranes and enhances acyl chain disruption of PC/cholesterol. The correlation between the antibacterial activity and ability to depolarize the transmembrane potential of E. coli spheroplasts, as well as the ability to induce calcein release from vesicles, suggests that the bacterial membrane is their target. The data demonstrate that preassembly of cationic diastereomeric antimicrobial peptides is an essential factor in their membrane targeting.     

HP(2-9)-magainin 2(1-12), a synthetic hybrid peptide, exerts its antifungal effect on Candida albicans by damaging the plasma membrane.

In our previous study, HP(2-9)-MA(1-12), HP-MA for short, a hybrid peptide incorporating residues 2-9 of Helicobacter pylori ribosomal protein L1 (HP) and residues 1-12 of magainin 2 (MA) was shown to have strong antibacterial activity. In this study the antifungal activity of HP-MA was evaluated using various fungi, and it was shown that the activity was increased when compared with the parent peptides. In order to investigate the fungicidal mechanism(s) of HP-MA its action against fungal cell membranes was examined by the potassium-release test, which showed that HP-MA caused an increase in the amount of K+ released from the cells. Furthermore, HP-MA induced significant morphological changes. These facts suggested that the fungicidal effect of HP-MA involves damaging the fungal cell membranes. CD investigators suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect, but that alpha-helicity is less directly correlated with the enhanced antibiotic activity of the hybrid.     

家蝇幼虫血淋巴中抗真菌肽的诱导方法比较及抗真菌活性

以未诱导组作为空白对照研究比较真菌诱导、超声诱导和热诱导家蝇Musca domestica 幼虫血淋巴初提液的抗真菌肽效果,比较各种诱导方法诱导后的幼虫存活率;用凝胶层析法和高效液相分离纯化热诱导家蝇3龄幼虫抗真菌肽,检测其抗白假丝酵母菌Candida albicans和新生隐球菌Cryptococcus neoformans活性;SDS-PAGE分析抗真菌肽的蛋白分子量范围。结果表明：3种诱导方法诱导后家蝇幼虫均产生具有明显抗真菌作用的抗真菌肽,其初提液抑菌圈大小没有明显差别;真菌诱导组和热诱导组幼虫存活率低于对照组,而超声诱导组与对照组相比则无明显差别。经分离纯化后,抗真菌肽仍具有较好的抗真菌活性;SDS-PAGE分析表明该抗真菌肽有效成分的蛋白分子量在14.4 kD以下。结果提示热诱导家蝇幼虫产生抗真菌肽是一种方便、有效的诱导方式。     

Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani

AIM: To identify antimicrobial peptides with high lytic activity against Rhizoctonia solani strain LR172, causal agent of rice sheath blight and aerial blight of soyabeans in the US. METHODS AND RESULTS: Among 12 natural and synthetic antimicrobial peptides tested in vitro, the wheat-seed peptide, purothionin, showed the strongest inhibitory activity that was similar to the antifungal antibiotics, nystatin and nikkomycin Z. Cecropin B, a natural peptide from cecropia moth, and synthetic peptide D4E1 produced the highest inhibitory activity against R. solani among linear peptides. Membrane permeabilization levels strongly correlated with antifungal activity of the peptides. Noticeable changes in membrane integrity were observed at concentrations of >/=0.5 micromol l(-1) for purothionin, 2 micromol l(-1) for cecropin B, D4E1, D2A21, melittin, and phor21, and 8 micromol l(-1) for magainin II and phor14. An increase of nuclear membrane permeabilization was observed in fungal cells treated with cecropin B, but not with purothionin. Diffusion of nuclear content was observed by fluorescent microscopy 10 min after adding a lethal concentration of cecropin B. Evaluation by electron microscopy confirmed severe cytoplasmic degradation and plasma membrane vesiculation. Purothionin and cecropin B were the most stable against proteolytic degradation when added to liquid cultures of R. solani. CONCLUSIONS: Purothionin, cecropin B, D4E1 and phor21 were shown to exhibit high in vitro lytic activity against R. solani strain LR172 for rice and soyabean. These peptides are greater than 16 amino acids long and rapidly increase fungal membrane permeabilization. Resistance to proteolysis is important for sufficient antifungal activity of antimicrobial peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected antimicrobial peptides offer an attractive alternative to traditional chemicals that could be utilized in molecular breeding to develop crops resistant to rice sheath blight and aerial blight of soyabean.     

Combinatorial approach to lead optimization of a novel hexapeptide with antifungal activity

Three sets of sublibraries of an antifungal lead peptide His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH2 (I) have been prepared by introducing variations at positions 1, 4 and 6. They were screened for their antifungal activity against C. albicans and C. neoformans in order to quantify inhibition at each step of the hexapeptide sublibrary iteration. The studies led to the identification of Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH2 as a novel hexapeptide with potent antifungal activity against both C. albicans and C. neoformans.     

三条新颖短肽筛选及抑菌作用的初报

[目的]为了发现新的农作物病原菌抗菌肽,人工设计并构建了大容量短肽文库,从中筛选并合成96条短肽用于鉴定其对农作物病原菌的抑菌活性.[方法]采用琼脂扩散法,对靶标菌一棉花枯萎病菌(Fusarium f.sp.vasinfecum)、棉花红腐病菌(Fusarium moniliforme)、小麦根腐病菌(Bipolaris sorokiniana)和马铃薯早疫病菌(Alternaria solani)进行抑菌初筛,并测定了有抗菌作用短肽的最小抑菌浓度和抑菌持久性.[结果]得到了A6、D4和F10对上述四种病原真菌抑菌效果较强,抑菌时间较长的抗菌肽,通过与抗菌肽数据库氨基酸序列对比,未见这3条抗菌肽的同源序列.[结论]研制的3条短肽属于新颖抗菌肽,为防治农作物真菌病害提供了新的基因资源.     

Structure-activity relationship study: short antimicrobial peptides.

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.     

Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice

Cryptococcus neoformans infection is a common fungal infection in persons infected with human immune deficiency virus (HIV) or those with defective cell-mediated immunity. Since treatment of cryptococcal meningitis poses a big challenge, the present study aimed to develop a novel liposomal therapeutic formulation against cryptococcosis. Treatment with tuftsin-incorporated liposomes increased the anti-cryptococcal activity of murine peritoneal macrophages. Prophylactic treatment of mice with tuftsin-incorporated liposomes reduced the dissemination of C.?neoformans to brain tissues. Moreover, the co-administration of tuftsin with nystatin liposomes augmented the anti-cryptococcal activity of nystatin, as mice treated with tuftsin-incorporated nystatin liposomes showed the highest survival and least fungal burden in their brain tissues. The results of the present study favour the use of immune-stimulating molecules along with antifungal agents in the treatment of opportunistic fungal infections.     

Influence on the plasma membrane of Candida albicans by HP (2-9)-magainin 2 (1-12) hybrid peptide

A 20-residue hybrid peptide (HP (2-9)-MA (1-12): HP-MA), incorporating 2-9 residues of Helicobacter pyroli ribosomal protein L1 (HP) and 1-12 residues of magainin 2 (MA), has more potent antibacterial activity than parent peptide HP (2-20) and magainin 2. In this study, the antifungal activity and its mechanism of HP-MA were investigated. HP-MA displayed a strong antifungal activity in an energy-dependent manner. To elucidate the antifungal mechanism(s) of HP-MA, FACScan analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe of Candida albicans were examined. The results indicated that the HP-MA exerts its antifungal effect by acting on the plasma membrane. Furthermore, the peptide induced remarkable morphological change when tested for membrane disrupting activity using liposomes (PC/Cholesterol; 10:1, w/w). In C. albicans, dimorphism plays a crucial role in pathogenesis but HP-MA could disrupt the mycelial forms and exert its antifungal effect on the blastoconidia in 20% fetal bovine serum.     

Synthesis and antimicrobial studies of novel 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one 4'-phenylthiosemicarbazones

New series of 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one 4'-phenylthiosemicarbazones (compounds 9-16) was obtained from the corresponding 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ones. The synthesized compounds have been characterized by their elemental, analytical, and spectral studies. Besides, these reported compounds were screened for their antibacterial and antifungal activities against a spectrum of microbial organisms. These studies proved that against bacteria, compounds 10 and 11 against Bacillus subtilis, compound 13 against Salmonella typhi, show maximum inhibition potency at low concentration (6.25 μg/mL), whereas against fungal, compounds 11, 13, and 16 against Candida albicans and compounds 12 and 13 against Cryptococcus neoformans, showed beneficial antifungal activity at minimum concentration (6.25 μg/mL).     

Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus

AIMS: In a search for an antifungal peptide with a high activity against Aspergillus flavus, Bacillus subtilis AU195 was selected from a collection of isolates with antagonistic activity against A. flavus. METHODS AND RESULTS: To identify the antifungal peptides, a protein purification scheme was developed based on the detection of the antifungal activity in purified fractions against A. flavus. Two lipopeptides were purified with anion exchange and gel filtration chromatography. Their masses were determined to be 1045 and 1059 m/z with mass spectrometry, and their peptide moiety was identical to bacillomycin D. CONCLUSION: AU195 synthesized a mixture of two antifungal bacillomycin D analogues with masses of 1045 and 1059, the 14 mass unit difference representing the difference between a C15 and a C16 lipid chain. SIGNIFICANCE AND IMPACT OF THE STUDY: Both bacillomycin D analogues were active at the same concentration against A. flavus, but the different lipid chain length apparently affected the activity of the lipopeptide against other fungi.     

Synthesis of (1,4)-naphthoquinono-[3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives as antifungal, antibacterial, and anticancer agents

A series of (1,4)-naphthoquinono [3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives 2-7 were synthesized and evaluated for antifungal, antibacterial, and anticancer activities. The structure-activity relationship of these compounds was studied and the results show that the compound 2b exhibited in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, and also possessed antibacterial profile against Klebsiella pneumoniae and Escherichia coli whereas 1c showed anticancer activity against Walker 256 Carcinosarcoma in rats.     

A tridecapeptide possesses both antimicrobial and protease-inhibitory activities

A 13-residue synthetic peptide (Rev4) was designed based on indolicidin, an antimicrobial peptide from bovine neutrophils. The synthetic peptide retains high antimicrobial activity. When tested for its stability in tobacco leaf extracts, Rev4 was highly stable compared to another antimicrobial peptide, magainin. When mixed with Rev4, magainin was protected from degradation by the leaf extract. Our results show that Rev4 is a potent protease inhibitor which selectively inhibits three out of four different types of proteases. Four other synthetic peptides were tested and the results were suggestive of no correlation between their antimicrobial and protease inhibitory activities.     

Effect of drastic sequence alteration and D-amino acid incorporation on the membrane binding behavior of lytic peptides

The amphipathic alpha-helix is a common motif found in many cell lytic peptides including antimicrobial peptides. We have recently shown that significantly altering the amphipathic structure of a lytic peptide by reshuffling its sequence and/or replacing a few l-amino acids with their D-enantiomers did not significantly affect the antimicrobial activity of the peptides nor their ability to bind and permeate negatively charged (PE/PG) membranes. However, a pronounced effect was observed regarding their hemolytic activity and their ability to bind and permeate zwitterionic (PC/Cho) membranes. To shed light on these findings, here we used surface plasmon resonance (SPR) with mono- and bilayer membranes. We found that the L-amino acid (aa) peptides bound 10-25-fold stronger to PC/Cho bilayers compared with monolayers, whereas the diastereomers bound similarly to both membranes. A two-state reaction model analysis of the data indicated that this difference is due to the insertion of the L-aa peptides into the PC/Cho bilayers, whereas the diastereomers are surface-localized. In contrast, only an approximately 2-fold difference was found with negatively charged membranes. Changes in the amphipathicity markedly affected only the insertion of the L-aa peptides into PC/Cho bilayers. Furthermore, whereas the all-L-aa peptides bound similarly to the PC/Cho and PE/PG membranes, the diastereomers bound approximately 100-fold better to PE/PG compared with PC/Cho membranes, and selectivity was determined only in the first binding step. The effect of the peptides on the lipid order determined by using ATR-FTIR studies supported these findings. Besides shedding light on the mode of action of these peptides, the present study demonstrates SPR as a powerful tool to differentiate between non-cell-selective compared with bacteria-selective peptides, based on differences in their membrane binding behavior.     

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.     

Synthesis of thiol-reactive lipopeptide adjuvants. Incorporation into liposomes and study of their mitogenic effect on mouse splenocytes

Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of importance for the further optimization of antigen-specific liposomal-based synthetic vaccines; the bromoacetyl derivative of Pam(3)CAG should be a promising lipopeptide derivative serving as an anchor for peptide epitopes while retaining its lymphocyte activation activity.     

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.     

Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides

Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.